Antibodies to a novel mammalian protein associated with uncontrolled cell division

ABSTRACT

Three mammalian nuclear proteins are disclosed which are useful in the diagnosis and prognosis of tumors of lymphoid and epithelial origin. The three proteins are immunologically related to each other. The level of expression of the proteins correlates with the malignant potential of lymphoid and epithelial tumors. In addition, in some cases the subcellular location of the proteins is indicative of malignant potential. Antibodies reactive with the proteins are disclosed as diagnostic tools, as are nucleic acid probes and primers for quantitating the messenger RNAs encoding the proteins. Methods for preparing and purifying the proteins are also taught.

The work leading to this invention was supported in part by Grant No.RO1CA54404 from the National Institutes of Health. The U.S. Governmentretains certain fights in this invention.

This application is a continuation-in-part of U.S. Ser. No. 07/995,930,filed Dec. 24, 1992, now abandoned, which is a continuation of U.S. Ser.No. 07/561,989, filed Aug. 1, 1990, now abandoned.

BACKGROUND OF THE INVENTION

A number of proliferation-associated nuclear proteins have beendescribed (Wheely and Baserga, 1977, Cell Biol. Int. Rep., vol. 1, p.13-21; Tan et al., 1987, Nucleic Acids Res., vol. 15, pp. 9299-9308;Gomez-Marquez et al., 1989, J. Biol. Chem., vol. 264 pp. 8451-8454;Feuerstein et al., 1988, J. Cell Biol., vol. 107, pp. 1629-1642; Shawyeret al., 1989, J. Biol. Chem., vol. 264, pp. 1046-1050; Jaskulski et al.,1988, J. Biol. Chem., vol. 263, pp. 10175-10179). Some, such asproliferating cell nuclear antigen (Tan et al., 1987, Nucleic AcidsRes., vol. 15, pp. 9299-9308; Jaskulski et al., 1988, J. Biol. Chem.,vol. 263, pp. 10175-10179), a co-factor of DNA polymerase delta,participate directly in proliferation. Still others, such as KI-67, areof unknown function (Gerdes et al., 1984, J. Immunol. vol. 133, pp.1710-1715). In cases involving nuclear phosphoproteins, phosphorylationand dephosphorylation through systems of kinases and phosphatases may beimportant in coordinating molecular functions (Morla et al., 1989, Cell,vol. 58, pp. 193-203; Chen et al., 1989, Cell, vol. 58, pp. 1193-1198;Cooper and Whyte, 1989, Cell, vol. 58, pp. 1009-1011). While theseproteins are expressed under normal physiological conditions, there isthe possibility that derangement of the expression of one or more ofthese proteins could be involved in the growth and development ofmalignancies.

There is a continuing need in the medical arts for new means to diagnoseand prognose cancers. Quick and simple methods can lead to morewidespread cancer testing and earlier diagnoses, which can save lives byallowing therapy at earlier stages of the disease process.

SUMMARY OF THE INVENTION

It is an object of the invention to provide substantially purifiedpreparations of mammalian proteins which are diagnostic and prognosticof human cancers.

It is another object of the invention to provide preparations ofantibodies which are immunoreactive with mammalian proteins and whichare useful in the diagnosis and prognosis of human cancers.

It is yet another object of the invention to provide methods ofproducing and purifying mammalian proteins which are diagnostic andprognostic of human cancers.

It is an object of the invention to provide diagnostic methods forpredicting malignant potential of lymphoid and epithelial tumors.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment asubstantially purified preparation of mammalian protein is providedwhich: has a molecular weight of about 35 kD; binds to myosin filaments;and is a substrate for casein kinase II in vitro.

In another embodiment of the invention substantially purifiedpreparations of a mammalian protein are provided which protein has amolecular weight of about 32 kD and is immunoreactive with antibodiesdirected against pp35; and is a substrate for casein kinase II in vitro.

In still another embodiment of the invention substantially purifiedpreparations of a mammalian protein are provided which protein has amolecular weight of about 42 kD and is immunoreactive with antibodiesdirected against pp35.

In another embodiment of the invention a preparation of antibodies isprovided which is immunoreactive with a native mammalian protein which:has a molecular weight of about 35 kD; binds to myosin filaments; and isa substrate for casein kinase II in vitro.

In still another embodiment of the invention a preparation of antibodiesis provided which is immunoreactive with a mammalian protein which: hasa molecular weight of about 32 kD; is immunoreactive with antibodiesdirected against pp35; and is a substrate for casein kinase II in vitro.

In still another embodiment of the invention a preparation of antibodiesis provided which is immunoreactive with a mammalian protein which: hasa molecular weight of about 42 kD; and is immunoreactive with antibodiesdirected against pp35.

In yet another embodiment a preparation of antibodies is provided whichis immunoreactive with a polypeptide comprising amino acid sequences asshown in FIG. 9.

In still another embodiment of the invention a preparation of antibodiesis provided which is produced by immunizing animals with an immunogencomprising a polypeptide comprising amino acid sequences as shown inFIG. 9.

In another embodiment a method is provided of purifying mammalianproteins pp35 and pp32 comprising: lysing cells in a detergent andlow-ionic strength buffer to form a cell lysate; separating componentsof the cell lysate by DEAE-cellulose chromatography and selectingdesired components; separating the desired components into fractions byHPLC anion-exchange chromatography and selecting desired fractions;separating the desired fractions into constituents by HPLChydroxylapatite chromatography and selecting desired constituents,desired constituents having a molecular weight of about 35 or about 32kD.

In still another embodiment of the invention, nucleic acid primers areprovided for amplifying sequences encoding pp32, pp35 or pp42.

In another embodiment of the invention a nucleic acid probe is providedwhich is complementary to mRNA encoding pp32, pp35 or pp42.

In still another embodiment of the invention diagnostic methods areprovided for predicting malignant potential of lymphoid and epithelialtissues comprising: providing a section of human lymphoid or epithelialtissue; determining levels of or intracellular sites of expression of agene product expressed from a gene selected from the group consistingof: pp32, pp35, and pp42.

In yet another embodiment of the invention a method of producing apreparation of a mammalian protein selected from the group consisting ofpp32, pp35, and pp42 is provided comprising: culturing a mammalianlymphoblastoid cell line; collecting the mammalian protein from thenucleus of cells of the cell line.

In still another embodiment of the invention, a method is provided fortreating tumors characterized by increased expression of pp32, byablation of pp32 which will either directly result in cell death or willpotentiate the effects of chemotherapeutic agents that ultimately killcells through programmed cell death. In particular, the inventionprovides a method for inhibiting proliferation of cells having potentialfor continuous increase in cell number (e.g., stem cells or neoplasticcells) by obtaining a DNA molecule comprising a cDNA sequence operablylinked to a promoter such that it will be expressed in antisenseorientation, the cDNA having all or part of the sequence of pp32, andtransfecting, with the DNA molecule, the cells with potential foruncontrolled proliferation.

In yet another embodiment of the invention, a method is provided forscreening candidate drugs to detect drugs with potential for decreasingthe rate of accumulation of tumor cells by incubating the candidate drugwith cells transfected with DNA encoding pp32 and monitoring one or morebiological activities of pp32 in the transfected cells. Particularlypreferred activities of pp32 for monitoring in such a screening assayinclude inhibition of programmed cell death, inhibition ofco-transformation by two oncogenes, such as ras and myc, or induction ofmalignant nuclear morphology in the transformed cell. These and otherembodiments of the invention will be described in more detail below.

The present invention thus provides the arts of oncology and pathologywith entirely new diagnostic tools for determining the malignantpotential of lymphoid and epithelial tumors. Further, by disclosing thediscovery of a protein (pp32) that functions in the pathway by whichcontrol of proliferation is released, the invention provides a newtherapeutic target for tumor therapy as well as methods to aid inselecting drugs specific for cells characterized by uncontrolledproliferation (i.e., neoplastic cells). The human nucleic acid sequenceencoding pp32 provided herein may be used for more specific diagnosticassays of tumor tissue or in the creation of antisense expressionvectors to inhibit the expression of pp32 by tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows myosin-affinity separation of pp35. A lysate prepared fromA₂₀ cells (8 mg total protein) was incubated with 1 mg of cross-linkedmyosin for 30 min at 4°. The myosin was pelleted at 16,000×g for 10min., the supernatant removed, and the pellet washed three times inlysis buffer. The pellet was then resuspended in lysis buffer containing1M KCl and incubated at 4° for 15 min. After incubation, the myosin wasagain pelleted. The figure represents total A₂₀ lysate, lane A; myosinprecipitate, lane B; myosin supernatant, lane C; KCl myosin precipitate,lane D; and KCl myosin extract, lane E. The figure shows aCoomassie-stained 7-15% gel, Myosin pellets were prepared forelectrophoresis using Laemmli solubilizing buffer withoutβ-mercaptoethanol to avoid solubilizing the disulfide cross-linkedmyosin; β-mercaptoethanol was added after re-centrifugation and prior toelectrophoresis.

FIG. 2 shows detection of pp35 and pp32 by antibody to denatured pp35(pp35d). The figure represents a Western blot of A₂₀ cell lysatedeveloped using anti-pp35d and ¹²⁵ I-protein A.

FIG. 3 shows analysis of pp42, pp35, and pp32 by peptide mapping. pp42,pp35, and pp32 were each excised from Coomassie-stained gels, iodinated,digested with α-chymotrypsin, applied to cellulose sheets, and subjectedto high-voltage electrophoresis along the horizontal dimension, and tothin-layer chromatography in the vertical dimension. The figurerepresents the resulting autoradiographs. The top row shows the map ofeach protein individually, while the bottom row illustrates a mixingexperiment in which equal amounts of radioactivity of the indicatedprotein digests were mixed and mapped together. In the mixingexperiment, co-migrating peptides appear at full intensity, whilepeptides contributed by only one of the proteins appear diminished inintensity.

FIG. 4A shows purification of pp35 and pp32. Panel A represents aCoomassie-stained gel of successive alternate fractions from theHPLC-anion exchange column. The figure shows the partial separation ofpp35, pp32, and pp42 from one another. Panels B and C representCoomassie-stained gels showing the homogeneous pp32 and pp35 obtainedafter HPLC hydroxylapatite chromatography. The numbered arrows indicatethe positions of molecular weight standards in kDa.

FIG. 4B shows immunoreactivity of partially-purified pp35, pp32, andpp42. Panel B illustrates a Western blot of an HPLC anion exchangefraction similar to those shown in FIG. 4A. Antibody to denatured pp35identifies three species, pp42, pp35, and pp32. Panel A is aCoomassie-stained gel lane showing purified pp35 and pp32, and isincluded as a standard. The numbered arrows indicate the positions ofmolecular weight standards in kDa.

FIG. 5 shows specificity of antibodies to native pp35 and pp32. Thefigure represents a Western blot of A₂₀ lysate using affinity-purifiedantibody to native pp32 (anti-pp32n) in lane B. Anti-pp35n reactsprimarily with pp35, but also slightly with pp32, pp42, and anunidentified band of approximately 68 kDa. Anti-pp32n reacts principallywith pp32, but cross-reacts slightly with pp35.

FIGS. 6A-6E show lipopolysaccharide stimulation of resting B cells.Purified small dense B cells were incubated with 40 μg/mllipopolysaccharide from E. coli 0127:B-8 under conditions whichconsistently yield a 100-fold stimulation of thymidine incorporationmeasured by 72 h. Aliquots were removed at times 0, 1 h, 24 h, 48 h, and72 h. The cell number and protein content of each aliquot wasdetermined, and a portion of each was analyzed by immunoblotting with acocktail of affinity-purified anti-pp35n and anti-pp32n and developingwith ¹²⁵ I-protein A. For quantitation, the experiment included astandard curve prepared with purified pp35 and pp32. The resultantautoradiographs (FIG. 6E) were quantitated by computerized densitometricimage analysis. FIGS. 6A-6D show the results for pp35 normalized tototal cell protein (FIG. 6A) and to cell number (FIG. 6C), and for pp32(FIGS. 6B and 6D). FIG. 6E shows the autoradiographs from which the datain FIGS. 6A-6D was obtained. Panel A represents the standard curve.Beginning at the left, the first pair of lanes illustrates the duplicatedeterminations for 31 ng each of pp35 and pp32. Each successive pair oflanes represents 62.5, 125, 250, and 500 ng. Panel B show theexperimental autoradiographs. Beginning at the left, the first pair oflanes represents duplicate determinations for the 72 h time point. Eachsuccessive pair of lanes represents duplicate determinations for the 48h, 24 h, 1 h, and 0 h time points.

FIGS. 7A and 7B show expression of pp35 and pp32 in cell lines. Cellsfrom the indicated lines were processed for quantitative immunoblottingas described in the description of FIG. 6. Equal amounts of cellularprotein from each line were analyzed. The figures show the mean ofduplicate determinations, and the error bars indicate the range; only asingle determination was available for the MOPC 21 subclone(P3.6.2.8.1). FIGS. 7A and 7B show the results respectively for pp35 andpp32. ABE-8.1/2 is a pre-B cell line. A₂₀, 2PK-3, and BCL₁ are all Bcell lines. MOPC 21 is a plasmacytoma.

FIG. 8 shows that pp35 and pp32 are phosphoproteins. A₂₀ cells werelabeled for 4 h by incubation with ³² P orthophosphate in otherwisephosphate-free medium. pp35 and pp32 were isolated as described,electrophoresed on a 10% Laemmli gel, transferred to nitrocellulose, andanalyzed by reactivity with antibodies to pp35 and pp32 (Lanes A) and byautoradiography (Lanes B). Antibody reactivity was detectedcolorimetrically.

FIG. 9 shows pp32-related cDNA sequences (SEQ ID NO: 4). Three hundredbases of sequence from the open reading frame of cloned pp32 cDNA areshown together with the predicted amino acid sequence. The approximately1 kb cDNA was subcloned into Bluescript and sequenced by thedideoxynucleotide method; approximately two-thirds of the insert hasbeen sequenced to date. The underlined sequence exactly matches sequenceobtained independently from a pp35 tryptic peptide isolated by reversephase chromatography.

FIGS. 10A and C show the cDNA sequences of human and murine pp32, (SEQID NO: 1 and SEQ ID NO: 3), respectively; the clones used to derivethose sequences are diagrammed in FIG. 10B.

FIG. 11 shows that recombinant murine pp32 fragment is recognized byantibody to native murine pp32 on Western blot.

FIG. 12 depicts a Western blot on which native human pp32, recombinanthuman pp32, and murine pp32 co-migrate and react with anti-pp32antibodies.

FIG. 13 shows that the in vitro translation product of human pp32 cDNAco-migrates with murine pp32 on SDS-PAGE.

FIG. 14 is a cartoon showing the structure of human and murine pp32.

FIG. 15 shows expression of human and murine pp32 RNA analyzed byNorthern blot.

FIG. 16 shows Northern blots demonstrating the time course of pp32 RNAlevels during induced differentiation of HL-60 cells.

FIG. 17 shows the dose-dependent inhibitory effect of pp32 ontransformation of rat embryo fibroblasts by ras and myc.

FIG. 18 shows the time course of BC12 and pp32-mediated resistance todrug-induced programmed cell death.

FIG. 19 is a photomicrograph showing the effect of pp32 on nuclearmorphology.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is a finding of the present invention that the expression of certainrelated mammalian nuclear proteins can be used to predict the malignantpotential of lymphoid and epithelial tumors. Antibodies have been raisedagainst the native forms of these proteins and they have been used asimmunohistochemical reagents. The percentage of cells which stainpositive with the reagents, the intensity of staining, and in some casesthe location of the staining, correlates with the malignant potential ofthe lymphoid or epithelial tumors.

DEFINITIONS

In describing the present invention, the following terminology is usedin accordance with the definitions set out below.

Nucleic Acids

A "double-stranded DNA molecule" refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itsnormal, double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5' to 3' direction along the nontranscribed stand of DNA(i.e., the strand having a sequence homologous to the mRNA).

A DNA sequence "corresponds" to an amino acid sequence if translation ofthe DNA sequence in accordance with the genetic code yields the aminoacid sequence (i.e., the DNA sequence "encodes" the amino acidsequence).

One DNA sequence "corresponds" to another DNA sequence if the twosequences encode the same amino acid sequence.

Two DNA sequences are "substantially homologous" when at least about 85%(preferably at least about 90%, and most preferably at least about 95%)of the nucleotides match over the defined length of the DNA sequences.Sequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See e.g.,Maniatis et al., supra; DNA Cloning, vols. 1 and II supra; Nucleic AcidHybridization, supra.

"Recombinant DNA" is a DNA molecule which includes DNA sequencesobtained from two or more species.

A coding sequence is an in-frame sequence of codons that (in view of thegenetic code) correspond to or encode a protein or peptide sequence. Twocoding sequences correspond to each other if the sequences or theircomplementary sequences encode the same amino acid sequences. A "codingsequence" in association with appropriate regulatory sequences may betranscribed and translated into a polypeptide in vivo. A polyadenylationsignal and transcription termination sequence will usually be located 3'to the coding sequence. A "promoter sequence" is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3' direction) coding sequence. A coding sequence is"under the control" of the promoter sequence in a cell when RNApolymerase which binds the promoter sequence transcribes the codingsequence into mRNA which is then in turn translated into the proteinencoded by the coding sequence.

For purposes of this invention, a cell has been "transfected" byexogenous DNA when such exogenous DNA has been introduced inside thecell membrane. Exogenous DNA may or may not be integrated (covalentlylinked) to chromosomal DNA making up the genome of the cell. Inprocaryotes and yeast, for example, the exogenous DNA may be maintainedon an episomal element such as a plasmid. With respect to eukaryoticcells, a stably transfected cell is one in which the exogenous DNA hasbecome integrated into a chromosome so that it is inherited by daughtercells through chromosome replication. This stability is demonstrated bythe ability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the exogenousDNA.

A "clone" is a population of cells derived from a single cell or commonancestor by mitosis.

A "cell line" is a clone of a primary cell that is capable of stablegrowth in vitro for many generations.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

Vectors are used to introduce a foreign substance, such as DNA, RNA orprotein, into an organism. Typical vectors include recombinant viruses(for DNA) and liposomes (for protein). A "DNA vector" is a replicon,such as plasmid, phage or cosmid, to which another DNA segment may beattached so as to bring about the replication of the attached segment.

An "expression vector" is a DNA vector which contains regulatorysequences which will direct protein synthesis by an appropriate hostcell. This usually means a promoter to bind RNA polymerase and initiatetranscription of mRNA, as well as ribosome binding sites and initiationsignals to direct translation of the mRNA into a polypeptide.Incorporation of a DNA sequence into an expression vector at the propersite and in correct reading frame, followed by transfection of anappropriate host cell by the vector, enables the production of a proteinencoded by said DNA sequence.

An expression vector may alternatively contain an antisense sequence,where a small DNA fragment, corresponding to all or part of an mRNAsequence, is inserted in opposite orientation into the vector after apromoter. As a result, the inserted DNA will be transcribed to producean RNA which is complementary to and capable of binding or hybridizingto the mRNA. Upon binding to the mRNA, translation of the mRNA isprevented, and consequently the protein coded for by the mRNA is notproduced. Production and use of antisense expression vectors isdescribed in more detail in U.S. Pat. No. 5,107,065 and U.S. Pat. No.5,190,931, both of which are incorporated herein by reference.

A "DNA library" is a population of vectors which each contain a DNAcoding sequence for some protein. The population as a whole encodes alarge number of peptides, and the sequence for a particular one of thepeptides can be recovered from the library using an appropriatescreening procedure.

"Amplification" of nucleic acid sequences is the in vitro production ofmultiple copies of a particular nucleic acid sequence. The amplifiedsequence is usually in the form of DNA. A variety of techniques forcarrying out such amplification are described in a review article by VanBrunt (1990, Bio/Technol., 8:291-294).

Polypeptides

Polypeptides are polymers made up of a sequence of amino acids linked bypeptide bonds, containing at least 10 and usually 50 or more amino acidsin the sequence. Proteins are polypeptides which usually have 35 or moreamino acids and form a characteristic three dimensional structure(tertiary structure).

Two amino acid sequences are "substantially homologous" when at leastabout 90% of the amino acids match over the defined length of the aminoacid sequences, preferably a match of at least about 92%, morepreferably a match of at least about 95%.

One amino acid sequence "corresponds" to another amino acid sequence ifat least 75% of the amino acid positions in the first sequence areoccupied by the same amino acid residues in the second sequence.Preferably 90% of the amino acid positions are identical, and mostpreferably 95% of the amino acid positions are identical. Alternatively,two amino acid sequences are considered to correspond to each other ifthe differences between the two sequences involve only conservativesubstitutions.

"Conservative amino acid substitutions" are the substitution of oneamino acid residue in a sequence by another residue of similarproperties, such that the secondary and tertiary structure of theresultant peptides are substantially the same. Conservative amino acidsubstitutions occur when an amino acid has substantially the same chargeas the amino acid for which it is substituted and the substitution hasno significant effect on the local conformation of the protein. Aminoacid pairs which may be conservatively substituted for one another arewell-known to those of ordinary skill in the art.

For the purposes of defining the present invention, two proteins arehomologous if 70% of the amino acids in their respective sequences arethe same; usually the amino acid sequences of homologous proteins are80% identical. The sequences of substantially homologous proteins willbe 85% identical, preferably the identity will be 90%, most preferably95%. Two proteins are similar if the majority of the differences betweentheir respective amino acid sequences involve conservativesubstitutions.

The polypeptides of this invention encompass pp32 and pp32 analogs. pp32is a naturally occurring, mature protein from mammals, especially miceand humans, and further encompasses all precursors and allelicvariations of pp32, as well as including forms of heterogeneousmolecular weight that may result from inconsistent processing in vivo.An example of the pp32 sequence is shown in FIG. 10A and SEQ ID NO: 2."pp32 analogs" are a class of peptides which includes:

1) "pp32 muteins," which are polypeptides which are substantiallyhomologous to pp32. It is sometimes preferred that any differences inthe amino acid sequences of the two proteins involve only conservativeamino acid substitutions. Alternatively, changes such as the eliminationof cysteine which alter the activity or stability of the protein may bepreferred.

2) "Truncated pp32 peptides," which include fragments of either "pp32"or "pp32 muteins" that preferably retain either (i) an amino acidsequence unique to pp32, (ii) an epitope unique to pp32 or (iii) pp32activity,

3) "pp32 fusion proteins" include heterologous polypeptides which aremade up of one of the above polypeptides (pp32, pp32 muteins ortruncated pp32 peptides) fused to any heterologous amino acid sequence.

"Unique" pp32 sequences, either amino acid sequences or nucleic acidsequences which encode them, are sequences which are identical to aportion of the sequence of a pp32 polypeptide, but which differ in atleast one amino acid or nucleotide residue from the sequences of pp35,and pp42, and preferably, are not found elsewhere in the human genome.Similarly, an epitope is "unique" to pp32 polypeptides if it is found onpp32 polypeptides but not found on any members of the homologous genefamily.

A composition comprising a selected component A is "substantially free"of another component B when component A makes up at least about 75% byweight of the combined weight of components A and B. Preferably,selected component A comprises at least about 90% by weight of thecombined weight, most preferably at least about 99% by weight of thecombined weight. In the case of a composition comprising a selectedbiologically active protein, which is substantially free ofcontaminating proteins, it is sometimes preferred that the compositionhaving the activity of the protein of interest contain species with onlya single molecular weight (i.e., a "homogeneous" composition).

As used herein, a "biological sample" refers to a sample of tissue orfluid isolated from a individual, including but not limited to, forexample, plasma, serum, spinal fluid, lymph fluid, the external sectionsof the skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, tumors, organs, and also samples of in vivocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents).

"Human tissue" is an aggregate of human cells which may constitute asolid mass. This term also encompasses a suspension of human cells, suchas blood cells, or a human cell line.

The term "coupled" as used herein refers to attachment by covalent bondsor by strong non-covalent interactions (e.g., hydrophobic interactions,hydrogen bonds, etc.). Covalent bonds may be, for example, ester, ether,phosphoester, amide, peptide, imide, carbon-sulfur bonds,carbon-phosphorus bonds, and the like.

An "epitope" is a structure, usually made up of a short peptide sequenceor oligosaccharide, that is specifically recognized or specificallybound by a component of the immune system. T-cell epitopes havegenerally been shown to be linear oligopeptides. Two epitopes correspondto each other if they can be specifically bound by the same antibody.Two antibodies correspond to each other if both are capable of bindingto the same epitope, and binding of one antibody to its epitope preventsbinding by the other antibody.

The term "immunoglobulin molecule" encompasses whole antibodies made upof four immunoglobulin peptide chains, two heavy chains and two lightchains, as well as immunoglobulin fragments. "Immunoglobulin fragments"are protein molecules related to antibodies, which are known to retainthe epitopic binding specificity of the original antibody, such as Fab,F(ab)'₂, Fv, etc.

Two polypeptides are "immunologically cross-reactive" when bothpolypeptides react with the same polyclonal antiserum or the samemonoclonal antibody.

General Methods

The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques are wellknown to the skilled worker and are explained fully in the literature.See, e.g., "DNA Cloning: A Practical Approach," Volumes I and II (D. N.Glover, ed., 1985); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);"Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins, eds., 1985);"Transcription and Translation" (B. D. Hames & S. J. Higgins, eds.,1984); "Animal Cell Culture" (R. I. Freshney, ed., 1986); "ImmobilizedCells and Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide toMolecular Cloning" (1984), Sambrook, et al., "Molecular Cloning: aLaboratory Manual" (1989) and "Current Protocols In Molecular Biology,"Ausubel, et al., eds., Current Protocols, 1994. Methods and discoveriesrelated to this invention are also disclosed in U.S. Application Ser.No. 07/561,989, filed Aug. 1, 1990, which is incorporated herein byreference.

DNA segments or oligonucleotides having specific sequences can besynthesized chemically or isolated by one of several approaches. Thebasic strategies for identifying, amplifying and isolating desired DNAsequences as well as assembling them into larger DNA moleculescontaining the desired sequence domains in the desired order, are wellknown to those of ordinary skill in the art. See, e.g., Sambrook, etal., (1989); B. Perbal, (1984). Preferably, DNA segments correspondingto pp32 may be isolated individually using the polymerase chain reaction(M. A. Innis, et al., "PCR Protocols: A Guide To Methods andApplications," Academic Press, 1990). A complete sequence may beassembled from overlapping oligonucleotides prepared by standard methodsand assembled into a complete coding sequence. See, e.g., Edge (1981)Nature 292:756; Nambair, et al. (1984) Science 223:1299; Jay, et al.(1984) J. Biol. Chem., 259:6311.

The assembled sequence can be cloned into any suitable vector orreplicon and maintained there in a composition which is substantiallyfree of vectors that do not contain the assembled sequence. Thisprovides a reservoir of the assembled sequence, and segments or theentire sequence can be extracted from the reservoir by excising from DNAin the reservoir material with restriction enzymes or by PCRamplification. Numerous cloning vectors are known to those of skill inthe art, and the selection of an appropriate cloning vector is a matterof choice (see, e.g., Sambrook, et al., incorporated herein byreference). The construction of vectors containing desired DNA segmentslinked by appropriate DNA sequences is accomplished by techniquessimilar to those used to construct the segments. These vectors may beconstructed to contain additional DNA segments, such as bacterialorigins of replication to make shuttle vectors (for shuttling betweenprokaryotic hosts and mammalian hosts), etc.

Procedures for construction and expression of mutant proteins of definedsequence are well known in the art. A DNA sequence encoding a mutantform of pp32 can be synthesized chemically or prepared from thewild-type sequence by one of several approaches, including primerextension, linker insertion and PCR (see, e.g., Sambrook, et al.).Mutants can be prepared by these techniques having additions, deletionsand substitutions in the wild-type sequence. It is preferable to testthe mutants to confirm that they are the desired sequence by sequenceanalysis and/or the assays described below. Mutant protein for testingmay be prepared by placing the coding sequence for the polypeptide in avector under the control of a promoter, so that the DNA sequence istranscribed into RNA and translated into protein in a host celltransfected by this (expression) vector. The mutant protein may beproduced by growing host cells transfected by an expression vectorcontaining the coding sequence for the mutant under conditions wherebythe polypeptide is expressed. The selection of the appropriate growthconditions is within the skill of the art.

According to the present invention, there are three related proteinswhich are expressed by mammalian lymphoblastoid cells as well as humanlymphoid and epithelial tumor cells. These proteins may be found intumor cells of epithelial tissues such as lung and bronchi and upperrespiratory system, liver, biliary, exocrine pancreas, head and necktissues including oropharynx, nasopharynx, and hypopharynx,gastrointestinal including esophageal, gastric, duodenal, jejunal, andcolorectal; endocrine including thyroid adrenal, endocrine pancreatic,parathyroid, and hypophyseal, genitourinary including kidney, bladder,prostrate, seminal vesicles, uterus, cervix, fallopian tubes, breast,skin and adenexal structures; tumor cells of lymphoid and hematopoietictissues including lymph nodes spleen, thymus, and bone marrow; tumorcells of mesenchymal tissues including skeletal muscle, cardiac muscle,smooth muscle, vascular tissues including endothelium, fibroconnectivetissue, adipose tissue; and in tumor cells of the central nervous systemtissues and peripheral nervous tissues. The proteins are relatedimmunologically and have also been shown to generate overlapping sets ofcleavage polypeptides. The proteins have been found to bephosphoproteins and are thus termed pp42, pp35, and pp32, based on theirmolecular weights. While the phosphoproteins were originally found inmouse cells, homologs have been found in human cells. The human homologsare immunologically cross-reactive with antibodies which are raisedagainst the murine proteins. Thus, such antibodies can be used in thepractice of the present invention's diagnostic method; alternativelyantibodies raised against the human homologs can be used. Antibodiesraised against synthetic or cleavage polypeptides can also be used.

As mentioned above the mammalian proteins of the present invention arebelieved to be phosphorylated. It has been found that two of theproteins, pp32 and pp35 are substrates for the enzyme casein kinase IIin vitro. Casein kinase II, or NII kinase (Rose et al., 1981, J. Biol.Chem., vol. 256, pp. 7468-7477), was initially described as a cyclicnucleotide-independent, heparin-sensitive kinase utilizing both ATP andGTP as phosphate donors; recent work suggests that there may be a familyof casein kinase II-like enzymes (Kishimoto et al., 1987, J. Biol.Chem., vol. 262, pp. 1344-1351). Several convergent lines of evidenceimply that casein kinase II plays a key role in the processes of cellproliferation and differentiation. Firstly, casein kinase II levels areelevated both in transformed cells (Brunati, et al., 1986, J. Immunol.vol. 127, pp. 2496-2501; Prowald, et al., 1984, FEBS Letters, vol. 174,pp. 479-483) and during embryogenesis (Perez, et al., 1987, Eur. J.Biochem., vol. 170, pp. 493-498; Schneider, et al., 1986, Eur. J.Biochem., vol. 170, pp. 733-738; additionally, casein kinase II levelsoscillate with the cell cycle (Carroll and Marshak, 1989, J. Biol.Chem., vol. 264, pp. 7345-7348) and undergo transient elevation duringcell differentiation (Sommercorn and Krebs, 1987, J. Biol. Chem., vol.262, pp. 3839-3843). Secondly, although casein kinase II substrates arenot limited to the nucleus (Hathaway and Traugh, 1982, Curr. top. Cell.Reg., vol. 21, pp. 101-127; Lees-Miller and Anderson, 1989, J. Biol.Chem., vol. 264, pp. 2431-2437; Wang, et al., 1986, Biochem. Biophys,Acta, vol. 888, pp. 225-236; Grande, et al., 1988, FEBS Letters vol.232, pp. 130-134), an ever-lengthening list of proteins which coordinatenuclear function are major substrates for casein kinase II (Matthews andHuebner, 1984, Mol. Cell. Biochem., vol. 59, pp. 81-99; Pfaff andAnderer, 1988, Biochem. Biophys. Acta, vol. 969, pp. 100-109) includingRNA polymerases I and II (Duceman, et al., 1981, J. Biol Chem., vol.256, pp. 10755-10758; Smiler and Rose, 1982, Biochemistry, vol. 21, pp.3721-3728), DNA topoisomerases I and II (Durban, et al., 1985, EMBO J.,vol. 4, pp. 2921-2926; Ackerman, et al., 1985, Proc. Natl. Acad. Sci.,USA vol. 82, pp. 3164-3168), high mobility group protein 14 (Walton, etal., 1985, J. Biol Chem., vol. 260, pp. 4745-4750) and C-proteins ofheterogenous nuclear ribonucleoprotein particles (Friedman, et al.,1985, Biochem. Biophys. Acta, vol. 847, pp. 165-176; Holcomb andFriedman, 1984, J. Biol. Chem., vol. 259, pp. 31-40). Thirdly, growthfactors such as insulin, epidermal growth factor, and insulin-likegrowth factor 1 stimulate casein kinase II activity in quiescent cells(Sommercorn, et al., 1987, Proc. Natl. Acad. Sci., USA, vol. 84, pp.8834-88389; Klarlund and Czech, 1988, J. Biol. Chem., vol. 263, pp.15872-15875; Ackerman and Osheroff, 1989, J. Biol. CHem., vol. 264, pp.11958011965). It appears that casein kinase II-mediated phosphorylationmust in some way modulate those cellular functions forming theinfrastructure of proliferation and differentiation. Whether pp42 isphosphorylated has not yet been determined, although pp42 is referred toherein as a phosphoprotein.

The 35 kD phosphoprotein of the present invention binds to myosinfilaments. This property can be used as a means of purifying thephosphoprotein from other cellular proteins. The other proteins of theinvention, of molecular weights 32 kD and 42 kD, do not bind to myosin,thus it appears that the myosin-binding domain is not present oraccessible in these proteins. However, as discussed below, peptidemapping has indicated that other regions of the 35 kD structure areshared by the 32 and 42 kD proteins. This is confirmed by the fact thataffinity purified antibodies which were raised against native pp35 areable to bind to both pp32 and murine pp42. No human homolog of murinepp42 has yet been observed.

According to the present invention, molecular weight is an importantidentifying property of the phosphoproteins. The molecular weights aredetermined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Suchtechniques are well known in the art. See Laemmli, 1970, Nature, vol.227, pp. 680-685.

The amino acid sequences of proteolytic peptides pp35 wereexperimentally determined. These exactly correspond to the predictedamino acid sequence of pp32. The predicted amino acid sequence of aportion of pp32 is shown in FIG. 9. This sequence or the sequences ofFIG. 10 can be used to synthesize polypeptides which can be used toraise antibodies as is known in the art. Alternatively, syntheticpolypeptides can be used (as can the whole protein) to compete withproteins present in a biological source for antibody binding. Suchcompetition can be used to assure that antibody binding is specific andcan also be used as a means of quantitating the amount of antigenpresent in a biological sample.

According to the present invention, the phosphoproteins can be isolatedfrom any convenient mammalian source. One particularly convenient sourceis a mammalian lymphoblastoid cell line. A particularly preferred cellline is the murine cell line A₂₀. See Kim, et al., 1979, J. Immunol.,vol. 129, pp. 549-554. This cell line is available from the AmericanType Culture Collection in Rockville, Md. Phosphoproteins pp35 and pp32can be collected form the nucleus of lymphoblastoid cell lines.Mammalian cell lines can be cultured according to methods known in theart using culture media and conditions which are well known.

Antibodies according to the present invention may be monoclonal orpolyclonal. If polyclonal antibodies are employed, it is preferred thatthey be affinity purified to render them monospecific. As mentionedabove, polyclonal antibodies which are raised against pp35 are alsoimmunoreactive with pp32 and pp42. Of course, since each of the proteinscontains unique peptide sequences, it is likely that monoclonalantibodies could be raised with would not cross-react with the othermembers of the phosphoprotein family. That is to say that the peptidemapping data make it likely that there are unique as well as sharedepitopes on each of the members of the phosphoprotein family.

Some antibodies according to the present invention are immunoreactivewith the native forms of the phosphoproteins. Thus, the proteins neednot be denatured in order to render them suitable binding partners forthe antibodies. However, the antibodies may be used to detect proteinsin the denatured form, such as in Western blots andimmunohistochemistry.

Other antibodies according to the invention are produced by immunizingexperimental, antibody-producing animals with an immunogen whichcomprises a polypeptide. The polypeptide contains amino acid sequencescorresponding to the phosphoproteins of the present invention, such asthose shown in FIG. 9. The immunogen may also contain other proteinssuch as keyhole limpet hemocyanin (KLH) which can be used to stimulatethe animal to produce an immune response to a polypeptide which may betoo small to do so on its own. Alternatively, the immunogen may compriseall or a portion of another protein which is attached to the polypeptideand produced from a fusion gene. Such fusions may be used to express thepolypeptide in bacterial or animal cells as is known in the art.

Methods have been developed to achieve preparations of apparenthomogeneity of the phosphoproteins of the present invention. The methodsinvolve lysis of the cells in a detergent and low-ionic strength buffer.Suitable detergents include Triton X-100 and other non-ionic detergents.The salt concentration of the buffers should preferably be in the rangeof 5-50 mM. Purification of a crude cell lysate can be accomplished bysequential chromatography on DEAE-cellulose, HPLC anion-exchange, andHPLC hydroxylapatite columns. Fractions which are eluted from thechromatographs can be assessed by SDS-PAGE and by Western blotting usinganti-pp35, for example. If desired an additional purification step canbe employed in the purification of pp35 which involves theco-precipitation with cross-linked myosin filaments. Rabbit skeletalmuscle is a suitable source of myosin. The phosphoproteins can be elutedfrom the myosin by extraction with 1M KCl.

A substantially purified preparation of mammalian protein according tothe present invention contains greater than about 75% of the desiredphosphoprotein. Preferably the preparation contains greater than about90% of the desired phosphoprotein. Most desirably the preparationcontains at least about 95% of the desired phosphoprotein.

Also contemplated by the present invention as a diagnostic andprognostic tool are nucleic acid probes. Like the antibodies of thepresent invention, they can be used to quantitate the expression of geneproducts from the genes encoding pp32, pp35, and pp42. Gene productsaccording to the invention include messenger RNA as well as protein orphosphoprotein. The probes are complementary to the mRNA sequences fromwhich the three phosphoproteins are translated. Probes may also be usedwhich vary slightly from the sequence of a particular phosphoproteingene. For example, a probe may be derived from the murine phosphoproteinsequence and be useful for hybridization to human and other mammalianspecies genes. Alternatively, a probe may represent an allelic orpolymorphic variant of a sequence. However, all probes must besufficiently homologous to the phosphoprotein gene being probed tohybridize under the conditions of the well known techniques of Northern,Southern, and in situ hybridization. Particularly preferred probes willhybridize under stringent conditions, e.g., washing in 0.2×SSC (0.03MNaCl+0.003M trisodium citrate) at 55° for Northern or 65° for Southernhybridization.

Nucleic acid probes are generally labeled with a radioactive moiety.Alternatively, they can be labeled with a fluorescent moiety or anenzyme which can cause a chromogenic substrate to change color. Nucleicacid probes are generally useful between about 15 and 1500 bases.Preferred probes usually contain 200-300 bases. Clones of the completecoding sequence may also be used. Such probes may be synthesized usingthe sequences disclosed in FIGS. 9 and 10. Alternatively, sequencesshown in FIGS. 9 and 10 can be used as a probe to detect other pp32sequences or pp35 or pp42 sequences. These latter sequences can be usedto derive probes for diagnostic uses.

Particularly preferred nucleic acid probes will have a sequence that isunique to pp32. Such sequences include sequences of at least 18consecutive nucleotides from FIG. 10, where the sequence preferablydiffers from the sequence of pp35 mRNA by at least 8 nucleotides withinthe contiguous sequence. As the expression of pp35, pp32 and pp42 hasbeen discovered to be elevated in relation to the degree of malignancyof a lymphoid or epithelial tumor, the steady state levels of thecorresponding mRNA are almost certainly elevated also in such tumors.Particular methods for quantitating mRNA in tissue samples are known inthe art, and any such method can be employed. It is possible that theelevated levels of the nuclear phosphoproteins of the invention are dueto gene amplification. The probes of the present invention can be usedin Southern hybridizations to detect such amplified genes.

Quantitative polymerase chain reaction (PCR) can be used to determinethe steady state amount of mRNA in a tissue. See Wang, et at., PNAS,vol. 86, pp. 9717-21, 1989. In order to practice this latter method toquantitate the amount of mRNA encoding the phosphoproteins of theinvention, primers are used which are derived from the phosphoproteingene sequences. As is known in the art, primers are complementary toopposite strands of a DNA duplex and flank a region of DNA to beamplified. To measure mRNA amounts cDNA is first made by reversetranscription and the cDNA is then amplified. PCR can also be used toquantitate genomic sequences which may be amplified in malignanttissues.

The diagnostic methods of the present invention are conveniently carriedout using standard histological sections, such as paraffin-embeddedsections. Either lymphoid or epithelial tissue can be used. Any of thepreparations of antibodies which are reactive with pp32, pp35 and/orpp42 can be used to immunostain the histological sections. Immunostainedsections can be analyzed to determine the percentage of cells whichimmunostain with the antibodies. As shown below in Table 1, the samplesderived from increasingly more malignant tissues showed increasingpercentages of immunostained cells. In the case of intermediate gradelesions, 60-70% of the cells are positive for the antibody stain. In thecase of high grade lesions, greater than 90% of the cells are positivefor the antibody stain.

Alternatively, in the case of staining with an anti-pp35 preparation,the localization of the immunostaining within the cells can bedetermined. Whereas in normal lymphoid tissue staining is restricted togerminal centers and small paracortical foci, in low grade lesions thestaining is more intense and more widely distributed. In intermediateand some high grade lesions (diffuse large cell malignant lymphoma(diffuse histiocytic lymphoma)) the staining is in both the nucleus andthe cytoplasm. In other high grade lesions (small non-cleaved cellmalignant lymphoma (diffuse undifferentiated lymphoma)) the staining issolely in the nucleus. In the case of pp32 staining, normal tissuesstain in the germinal centers and paracortex, whereas in the malignanttissues the staining is in the nucleus. Staining of pp32 does not appearin the cytoplasm in any of the lesions examined.

Cloning, analysis of pp32 sequence, and transfection studies haveprovided a major window into pp32 structure and function. pp32 cDNAencodes a 28.6 kDa protein (see e.g., SEQ ID NO: 2); the N-terminalapproximately two-thirds predicts an amphipathic alpha helix containingtwo possible nuclear localization signals and a potential leucine zippermotif. Preferable nucleotide probes are directed to this region (seeFIG. 10, bases 1-550) or to the 3' untranslated region (see FIG. 10,from base 842 to the end). The C-terminal third is exceptionally acidic,comprised of approximately 70% aspartic and glutamic acid residues; thepredicted pI of human pp32 is 3.81. Human and murine pp32 cDNA's are 88%identical; the predicted proteins are 89% identical and 95% similar.While superficially, pp32 structure might suggest a transcription factorwith leucine zipper and acidic domain, there is substantialcircumstantial evidence to the contrary: when expressed in normal cells,pp32 levels approach 10⁶ copies per cell; the half-life of pp32 proteinis approximately three days; pp32 does not bind DNA; and additionalfunctions have been identified for pp32.

In yet another embodiment of the present invention, the immunostainingcan be analyzed to determine the intensity of staining. Increasingintensity of staining correlates with increasing malignant potential.

Biological Activity and Therapy

pp32 is found in normal cells with stem cell properties and isoverexpressed in many neoplastic cells. For example, two related nuclearphosphoproteins, pp32 and pp35, have been identified in a murineneoplastic B-lymphoblastoid cell line A₂₀. The pattern of proteinexpression in vivo and in cell lines suggests a dual association: withself-renewing stem-like cell populations and also with neoplastic cells.Whereas the majority of the neoplastic tissue culture cells stainpositively for pp35 and pp32, the distribution in normal tissues ishighly restricted. Both intestinal crypt cells and basal cells ofsquamous epithelium stain positively with great specificity; these cellpopulations are well known to contain the cells which continually renewtheir respective tissues (i.e., stem cells). The initial antibodies tomurine pp32 and pp35 react well with their human counterparts.Additional small-scale studies, undertaken in prostate cancer andnon-Hodgkin's lymphoma, indicate that increased frequency and intensityof pp32 staining in human cancers correspond to increased malignantpotential.

Consistent with previous observations in vivo that pp32 is found inself-renewing cells but not in their terminally differentiated progeny,pp32 RNA levels are down-regulated during TPA-induced differentiation ofHL-60 cells. In co-transfection experiments, pp32 inhibited the abilityof ras and myc to transform rat embryo fibroblasts. AT3.1 rat prostaticcarcinoma cells stably transfected with human pp32 cDNA are resistant toprogrammed cell death induced by 5-fluorouracil, ionomycin, andthapsigargin. These results suggest that pp32 may play a key role inself-renewing cell populations where it may act to limit theirsensitivity to transformation and apoptosis.

Drug Screening Assays

Drugs which inhibit the biological activity of pp32 are good candidatesfor anti-tumor drugs, because they affect one of the steps that leads touncontrolled proliferation or a continuous increase in cell number.Therefore, the present invention provides a screening assay which willhelp identify anti-tumor drugs, when the results of this screening assayare considered in conjunction with the results from other model systems,such as in vivo tumor growth assays, clonagenic assays and in vitrocytotoxic tests. While any assay which tests inhibition of a biologicalactivity of pp32 may be used as a screening tool, three preferredscreens are described in greater detail below.

pp32 possesses three biologic activities which may be relevant both tostem cells and to cancer: (1) inhibition of co-transformation by twooncogenes, such as ras and myc; (2) partial protection againstprogrammed cell death; and (3) modulation of nuclear shape and size. Themolecular bases of these functions are completely unknown. Each of thesefunctions has been defined wholly or in part through transient or stabletransfection with pp32 expression constructs.

Stem cells, particularly those in renewing tissues must be resistant toneoplastic transformation. Indeed, special mechanisms have been proposedto preserve the integrity of stem cell populations (Cairns, J. (1975)"Mutation Selection and the Natural History of Cancer," Nature,255:197-200.). The relationship of cellular oncogenes to multi-stepcarcinogenesis was first demonstrated in 1983 by Land, Parada, andWeinberg (Land, et al. (1983) "Cellular Oncogenes and MultistepCarcinogenesis, " Science, 222:771-778; Land, et al. (1983) "TumorigenicConversion of Primary Embryo Fibroblasts Requires At Least TwoCooperating Oncogenes," Nature, 304:596-602) when they showed thatprimary cultures of rat embryo fibroblasts require both ras and myc fortumorigenic conversion. Six molecules have been subsequently shown toinhibit transformation by ras and myc in this system, although themechanisms are not fully understood: wild type p53 (Eliyahu, et al.,"Wild-type p53 can inhibit oncogene-mediated focus formation," Proc.Natl. Acad. Sci. USA, 86:8763-8767, 1989); c-jun (Ginsberg, et al.,"Transfected mouse c-jun can inhibit transformation of primary ratembryo fibroblasts," Oncogene, 6:669-672, 1991); B-myc (Resar, et al.,"B-Myc inhibits neoplastic transformation and transcriptional activationby c-Myc," Mol. Cell. Biol., 13:1130-1136, 1993.22); E1B type 5 (van denHeuvel, et al., "Large E1B proteins of adenovirus types 5 and 12 havedifferent effects on p53 and distinct roles in cell transformation," J.Virol., 67:5226-5234, 1993); hsc70, a rat heat-shock cognate (Yehiely,et al., "The gene for the rat heat-shock cognate, hsc70, can suppressoncogene-mediated transformation," Cell. Growth Diff., 3:803-809, 1992);and max (Makela, et al., "Alternative forms of max as enhancers orsuppressors of myc-ras cotransformation," Science, 256:373-377, 1992).We have discovered that pp32 is also one of the handful of moleculescapable of inhibiting ras-myc mediated transformation of rat embryofibroblasts. In normal cells, pp32 performs a critical function ofhelping to maintain stem cell integrity by suppressing transformingstimuli analogous to the ras-myc model.

Inhibition of Co-Transformation

Cell lines transfected with pp32 are resistant to co-transformation by acombination of two oncogenes or mutated tumor suppressor genes, forexample ras and myc, as described below in Example 17. Drug candidatesare considered positive if, when added to a ras-myc transformation assayalong with a pp32 expression vector, the drug stimulates an increase inthe number of transformed cells over the number observed for thetriply-transfected cells in the absence of the drug.

All measurements should preferably be performed at least in triplicateto permit standard deviations to be calculated; experiments willpreferably produce 75-100 colonies per flask in ras-myc controls inorder to be considered valid. (In the co-transformation experiment, mycmay be replaced by jun, adenovirus ELA or a mutant form of p53, oranother mutant tumor suppressor gene that gives comparable levels oftransformation in the controls. Similar substitution of ras with otheroncogenes is permissible, so long as controls show levels oftransformation comparable to those obtained with ras and myc.) As can beseen in FIG. 17, retention of full transformation inhibitory potencywill be obvious. The more difficult situation to assess will be partialinhibition. Reductions will usually have to achieve statisticalsignificance using a standard statistical measure such as a Student's ttest. Any untransformed cell line that is susceptible toco-transformation may be used in this assay. We have found primarycultures of rat embryo fibroblasts to be particularly preferable as thecell for transformation due to reduction in variability of the assay.

pp32 and Programmed Cell Death

The balance between proliferation and programmed cell death is thoughtto be a normal feature of tissue growth regulation (Gerschenson, et al.(1991) "Apoptosis and Cell Proliferation are Terms of the GrowthEquation," in Apoptosis: The Molecular Basis of Cell Death, J. Inglis,et al, eds., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, pp. 175-192; Kerr, et al. (1991) "Definition and Incidence ofApoptosis: An Historical Perspective," in Apoptosis: The Molecular Basisof Cell Death, J. Inglis, et al, eds., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, pp. 5-29. However, when abnormally low,programmed cell death can contribute to tumorigenesis throughinappropriate increases in cell number without increases inproliferative rate (Cope, et al. (1991) "Carcinogenesis and Apoptosis:Paradigms and Paradoxes in Cell Cycle and Differentiation," inApoptosis: The Molecular Basis of Cell Death, J. Inglis, et al, eds.,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp.61-86). Just as stem cell populations must preserve their integrity withrespect to transformation, they must also resist cell programmed celldeath.

Programmed cell death (Martin, et al. (1994) "Dicing With Death:Dissecting the Components of the Apoptosis Machinery," Trends Biochem.Sci., 19:26-30) occurs in a number of developmental and other settings.For example, when tissues involute due to withdrawal of hormonalstimulation, they do so through a process of programmed cell death(Buttyan, R. (1991) "Genetic Response of Prostate Cells to AndrogenDeprivation: Insights Into the Cellular Mechanism of Apoptosis," inApoptosis: The Molecular Basis of Cell Death, J. Inglis, et al, eds.,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp.157-173); only the stem cells persist to repopulate the tissue with thereturn of hormonal stimulation. Despite much study, the mechanismwhereby certain stimuli lead to characteristic morphologic changes andDNA fragmentation remain a subject of debate. Certain key observations,however, have been made. In appropriate growth-related stimuli can leadto programmed cell death; when myc is expressed in quiescent cells, suchas cells undergoing serum starvation, programmed cell death can result(Martin, et al., 1994; Wagner, et al. (1993) "Myc-Mediated Apoptosis isBlocked By Ectopic Expression of Bcl-2," Mol. Cell. Biol.,13:2432-2440).

BCL2 is a mitochondrial membrane (Hockenbery, et al. (1991) "BCL2Protein is Topographically Restricted in Tissues Characterized byApoptotic Cell Death," Proc. Natl. Acad. Sci., U.S.A., 88:6961-6965) andperhaps nuclear membrane (de Jong, et al. (1994) "SubcellularLocalization of the bcl-2 Protein in Malignant and Normal LymphoidCells," Cancer Res., 54:256-260) protein which protects againstprogrammed cell death, although the mechanism is unclear. BCL2 islocalized to stem cell areas in tissues where apoptotic death occurs,such as basal cells in squamous epithelium and intestinal crypt cells(Hockenbery, et al., 1991). BCL2 extends the lives of SF9 insect cellswhen overexpressed in baculovirus (Alnemri, et al. (1992) "OverexpressedFull-Length Human BCL2 Extends the Survival of Baculovirus-Infected Sf9Insect Cells," Proc. Natl. Acad. Sci., U.S.A., 89:7295-7299), andprotects against myc-mediated apoptosis (Wagner, et al., 1993).

pp32 also preserves stem cell integrity by contributing to resistance toprogrammed cell death, as shown in Example 18, below. Like BCL2, pp32both localizes to compartments where stem cells reside in vivo andprotects against at least one form of programmed cell death. pp32transfectants incubated in drug beyond 48 h will being to die offthrough an apoptotic mechanism, as confirmed by pulsed field gelelectrophoresis analysis of double-stranded DNA breaks. While continuedexpression of pp32 during extended drug treatment has yet to beverified, the data strongly suggest that pp32 and BCL-2 act throughdistinct mechanisms.

Protection from Programmed Cell Death

Assays of candidate drugs for interference with pp32-mediated inhibitionof programmed cell death will determine the relative protection forpp32-transfected cells achieved by the candidate drug at a fixed dose ofinducers of programmed cell death, such as 5-fluorouracil, ionomycin, orthapsigargin, as compared to BCL-2, and vector controls. (See Example 18for a typical procedure.) Data may be expressed as the ratio of theclonogenic potential of, for example, AT3.1 cells expressing pp32 in thepresence of the candidate to AT3.1 cells expressing pp32 in the absenceof the candidate drug. When differences are identified, they may beconfirmed by dose-response analysis, analysis of dependence on length ofdrug exposure, and analysis of DNA for double-stranded breaks. Theseconfirmatory studies should permit clear distinction among those drugswhich affect pp32-mediated protection from programmed cell death andthose which do not, since intact pp32 yields distinct dose-responsecurves, protects against double-stranded DNA breaks, and loses itsprotective effect after 48 h of drug exposure.

pp32 and Nuclear Grade

Nuclear morphology often undergoes profound change following malignanttransformation. Malignant nuclei are often misshapen, and the chromatinshows coarse areas of condensation and clearing, rather than the moreusual finely stippled pattern. Pathologists often make use of nucleargrading in an attempt to assess the degree of malignancy of a givenpatient's tumor. While nuclear grading is potentially useful, it can besubjective and prone to interobserver variation. In the early 1980's,nuclear roundness measurements were applied to human prostatic carcinomain an attempt to quantify nuclear grade through image analysis (Diamond,et al (1982) "A New Method To Assess Metastic Potential of HumanProstate Cancer: Relative Nuclear Roundness," J. Urol., 128:729-734;Diamond, et al. (1982) "Computerized Image Analysis of Nuclear Shape Asa Prognostic Factor for Prostatic Cancer," The Prostate, 3:321-332); theresults were that nuclear roundness measurements appears superior to theGleason grading system in at least one study assessing prostate cancer(Epstein, et al. (1984) "Nuclear Roundness Factor. A Predictor ofProgression in Untreated Stage A2 Prostate Cancer," Cancer,54:1666-1671). Subsequent studies extended the approach by applyingadditional nuclear measurements (texture, ellipicity, etc.) to renalcell cancer (Murphy, et al. (1990) "Nuclear Shape Analysis forAssessment of Prognosis in Renal Cell Carcinoma," J. Urol.,143:1103-1107), medullary carcinoma of the thyroid (Galera, et al.(1990) "Cytophotometric DNA Measurements in Medullary ThyroidCarcinoma," Cancer, 65:2255-2260), dysplastic nevi (Fleming, et al.(1990) "Image Analysis Cytometry of Dysplastic Nevi," J. Invest.Dermatol., 95:287-291), Wilm's tumor (Partin, et al. (1990) "NuclearMorphometry as a Predictor of Response to Therapy in Wilms Tumor: APreliminary Report," J. Urol., 144:952-954), breast cancer (Dawson, etal. (1991) "Nuclear Grading of Breast Carcinoma by Image Analysis.Classification by Multivariate and Neural Network Analysis,: Am. J.Clin. Pathol, 95:S29-S37; Pienta, et al. (1991) "Correlation of NuclearMorphometry with Progression of Breast Cancer," Cancer, 68:2012-2016),head and neck squamous cancers (Briggs, et al. (1992) "NuclearMorphometry for Prediction of Metastatic Potential in Early SquamousCell Carcinoma of the Floor of the Mouth," Arch. Otolaryngol Head. Neck.Surg., 118:531-533), colorectal cancer (Hill, et al. (1989) "TheProportion of Stem Cells in Murine Tumors," Int. J. Radiat. Oncol. Biol.Phys., 16:513-518), pancreatic cancer (Rickaert, et al. (1992)"Computerized Morphonuclear Characteristics and DNA Content ofAdenocarcinoma of the Pancreas, Chronic Pancreatitis, and NormalTissues: Relationship with Histopathologic Grading," Hum. Pathol.,23:1210-1215; Weger, et al. (1992) "Morphometry and Prognosis in Cancerof the Pancreatic Head," Pathol. Res. Pratt., 188:763-769), and ovariancancer (Drescher, et al. (1992) "Prognostic Significance of DNA Contentand Nuclear Morphology in Borderline Ovarian Tumors," Gynecol. Oncol.,48:242-246). Although the results were mixed, the overwhelming majorityof studies showed correlations of deteriorating nuclear morphology withincreasing malignancy.

Initial observations suggested that increases in pp32 content paralleledincreases in malignancy and, by extension, nuclear alternations but didnot suggest a causal relationship. Recombinant pp32 overexpression leadsto nuclear changes characteristic of high-grade malignancy includingincreased nuclear size with course areas of chromatin condensation andclearing.

Candidate drugs are screened by culturing cells transfected with pp32 inthe presence and absence of the drug, then comparing the nuclei of cellsin the two cultures. The effect of pp32 on nuclear morphology is usuallydetermined visually by direct observation of Papanicolaou-stained cellpreparations. Cultures wherein >60% of cells display nuclei withdiameters (measured with a reticle) >1.5 times the mean diameter ofcontrol cultures infected with wild-type baculovirus are typicallyconsidered positive for malignant nuclear morphology; such cells willalso display finely dispersed chromatin. The potential for subjectivitymay be addressed through quantitative image analysis and sizemeasurements (e.g. Coulter counter analysis of cells or isolatednuclei).

Therapy Based on pp32

Because of the profound clinical importance of prostate cancer, pp32expression has been studied in this disease. Prostate cancer cells areslow-growing and are thus relatively insensitive to agents which dependupon cell proliferation to exert their effects. In contrast, pp32 is notdirectly associated with proliferation, but rather with the failure ofprostate cancer cells to die; pp32 may thus address a major problem withandrogen ablation therapy, the frequent outgrowth ofandrogen-independent prostatic cancer cells.

The relationship between pp32 expression and self-renewing cellpopulations has been explored using androgen to manipulatehormonally-dependent rat tissues in a highly physiologic model in vivo.In the intact prostate, pp32 protein and mRNA were found primarily inthe peripheral regions thought to be roughly equivalent to intestinalcrypts in that they contain the self-renewing cell populations; overall,approximately 15% of epithelial cells stained positively for pp32. Incontrast, virtually all epithelial cells in the involuted prostate ofcastrated rats were positive for pp32 mRNA by in situ hybridization, andapproximately 55% were antibody-positive. Calculations of epithelialcell mass suggested that the residual pp32-positive population incastrates was roughly equivalent, numerically, to the pp32-positivepopulation in the intact gland. Simply stated, pp32 appeared to mark thestem-like cells capable of renewing the gland upon subsequent androgenstimulation. When androgen was administered to castrated rats, thepp32-positive cells appeared to dilute out to their original proportionand re-achieve their original anatomic distribution. Consistent with thedual finding of pp32 in stem-like self-renewing cell populations andcancer, pp32 levels and the number of pp32-positive cells increased withincreasing Gleason grade in human prostate cancer.

Androgen ablation is effective during an initial response period, whereandrogen dependent prostatic cancer cells die via programmed cell death.Several investigators have proposed potentiation of programmed celldeath as a therapeutic target. According to this scheme, programmed celldeath of otherwise resistant prostate cancer cells could be potentiatedeither by increasing entry into the pathway, or by interfering withendogenous mechanisms of resistance to apoptosis (programmed celldeath).

Ablation of pp32 in prostate cancer cell lines through knockoutmutations or through antisense approaches will either directly result incell death or will potentiate the effects of other agents. Manyconventional chemotherapeutic agents ultimately kill cells throughprogrammed cell death. Ablation of pp32, a molecule which confersresistance to programmed cell death, can therefore be reasonablyexpected to sensitize cells to conventional chemotherapeutic agents bypermitting increased entry into programmed cell death pathways. Therapywhich includes ablation of pp32 may be expected to be similarlyeffective in treatment of other tumors containing cells showingincreased pp32 expression.

Antisense Therapy

One approach to therapy of human cancer cells is to introduce vectorsexpressing antisense sequences to block expression of pp32. In oneembodiment of this invention, a method is provided for inhibitingproliferation of cells characterized by potential for continuousincrease in cell number, e.g., neoplastic cells, which comprisesobtaining a DNA expression vector containing a cDNA sequence having thesequence of human pp32 mRNA which is operably linked to a promoter suchthat it will be expressed in antisense orientation, and transforming theneoplastic cells with the DNA vector. The expression vector material isgenerally produced by culture of recombinant or transfected cells andformulated in a pharmacologically acceptable solution or suspension,which is usually a physiologically-compatible aqueous solution, or incoated tablets, tablets, capsules, suppositories, inhalation aerosols,or ampules, as described in the art, for example in U.S. Pat. No.4,446,128, incorporated herein by reference.

The vector-containing composition is administered to a mammal in anamount sufficient to transfect a substantial portion of the target cellsof the mammal. Administration may be any suitable route, including oral,rectal, intranasal or by intravesicular (e.g. bladder) instillation orinjection where injection may be, for example, transdermal,subcutaneous, intramuscular or intravenous. Preferably, the expressionvector is administered to the mammal so that the tumor cells of themammal are preferentially transfected. Determination of the amount to beadministered will involve consideration of infectivity of the vector,transfection efficiency in vitro, immune response of the patient, etc. Atypical initial dose for administration would be 10-1000 micrograms whenadministered intravenously, intramuscularly, subcutaneously,intravesicularly, or in inhalation aerosol, 100 to 1000 micrograms bymouth, or 10⁵ to 10¹⁰ plaque forming units of a recombinant vector,although this amount may be adjusted by a clinician doing theadministration as commonly occurs in the administration of otherpharmacological agents. A single administration may usually besufficient to produce a therapeutic effect, but multiple administrationsmay be necessary to assure continued response over a substantial periodof time.

Further description of suitable methods of formulation andadministration according to this invention may be found in U.S. Pat.Nos. 4,592,002 and 4,920,209, incorporated herein by reference.

The following examples are merely illustrative of the invention and donot limit the invention.

EXAMPLE 1

pp35 co-sediments with cross-linked myosin

pp35 co-sediments with rabbit skeletal muscle myosin filaments. Whilethis suggested a means of affinity purification, it required aconvenient means to separate pp35 from myosin. Since amine cross-linkersuniformly inactivated the ability of myosin to bind pp35, myosinfilaments were disulfide-cross-linked using o-phenanthroline and copper.Unlike native filaments, which disassemble in increasing saltconcentrations, the cross-linked filaments permitted recovery of pp35 byelution in 1M KCl. FIG. 1 Lanes B and C show the respective pellet andsupernatant resulting from incubation of cross-linked myosin in A₂₀lysate, while lanes D and E respectively show the residual pellet andthe material eluting from the myosin in 1M KCl. The eluate contained aCoomassie-stainable 35 kDa band. pp35 was further purified by HPLC anionexchange chromatography, excised from a Laemmli gel (Laemmli, 1970,Nature, vol. 227, pp. 680-685, and used to raise polyclonal antibodies,designated anti-pp35d (d designates that the immunogen was denatured).

Myosin Affinity Purification of pp35. Rabbit skeletal muscle myosin waspurified as described (Margossian, et al., 1982, Methods Enzymol., vol.895, pp. 55-71) using DEAE cellulose chromatography in pyrophosphatebuffers to remove the contaminating C-protein. For cross-linking,purified rabbit skeletal muscle myosin in 0.6M KCl, 50 mM potassiumphosphate, pH 6.5 at 15-20 mg/ml was diluted to 1 mg/ml in 10 mM sodiumphosphate, pH 7.5 and incubated at 4° for 15 min. After incubation, themixture was adjusted to 0.1 mg/ml myosin and brought to finalconcentrations of 1.7 mM (Cu(0-phenanthroline)₂, and 1% Triton X-100, pH7.5. Generally, a lysate prepared from A₂₀ cells (8 mg total protein,see below) was incubated with 1 mg of cross-linked myosin for 30 min. at4°. The myosin was pelleted at 16,000×g for 1 min., the supernatantremoved, and the pellet washed three times in lysis buffer. The pelletwas then resuspended in lysis buffer containing 1M KCl and incubated at4° for 15 min. After incubation, the myosin was again pelleted. Thematerial eluted from cross-linked myosin was further purified by DEAEcellulose chromatography. The 1M KCl extract was dialyzed into 20 mMKCl, 5 mM sodium phosphate, pH 7.6, with 0.1 mM henylmethylsulfonylfluoride, 1 mM 2-mercaptoethanol, and 0.1% Triton X-100. The extract wasloaded onto a 0.9×8 cm DEAE cellulose column equilibrated in the samebuffer, which was then undercut with 0.3M KCl and eluted with 0.5M KClin the same buffer. Following purification, the eluted material wasre-dialyzed into the starting buffer and stored at 4°. A typicalpreparation yielded approximately 100 μg of total product, includingimpurities, from approximately 2.5×10⁹ cells grown in 1 l of medium.

Antibody to denatured pp35 was raised in rabbits and affinity-purified.Initially, antigen was purified by electroelution fromSDS-polyacrylamide gels essentially as described (Knowles and Bologna,1983). To localize the band, 85 μg of pp35 kDa protein in 5 mM sodiumphosphate, 20 mM KCl, 0.1% Triton X-100, pH 8.5, was reacted with a100-fold molar excess of dansyl chloride. Derivatized protein was mixedwith unreacted 35 kDa protein at a 1:9 ration (w/w) and theelectrophoretic bands visualized under ultraviolet light. Protein waseluted from the gel slices using a commercial apparatus (Isco Corp.,Lincoln, Nebr.). Initially, each of the three female New Zealand whiterabbits was injected at six subcutaneous sites with 50 μg of 35 kDaprotein emulsified in phosphate-buffered saline containing 2% squalene,50 μg trehalose dimycolate and 100 μg monophosphoryl lipid A (RibiImmunochem Research, Inc., Hamilton, Mont.). Each rabbit received atotal volume of 1.8 ml. On day 19, the animals were boosted in similarfashion. On day 42, two of the rabbits were again boosted with 125 μg ofprotein directly excised from stained, neutralized gels and emulsifiedin the same adjuvant system. One rabbit responded, as determined bystrong reactivity against the homologous antigen in a Western blot. Thisanimal was bled on day 100. The resulting antiserum was purified on acolumn of pp35 kDa coupled to Reacti-Gel 6X (Pierce Chemical Co.,Rockford, Ill.) with the same elution protocol as described above.

EXAMPLE 2

Identification of pp42 and pp32

Anti-pp35d was affinity purified as described above and used to examineA₂₀ lysates. In immunoblots, affinity-purified anti-35d reacted wellwith pp35, and interestingly, with an additional 32 kDa proteindesignated pp32, as seen in FIG. 2. During fractionation, this antibodyalso detected an additional low-abundance 42 kDa species, designatedpp42 (FIG. 4, v.i.). Gel electrophoresis and Western blotting wereperformed as described. See Laemmli, 1970, Nature vol. 227, pp. 680-685;Towbin, et al., 1979, Proc. Natl. Acad. Sci., USA, vol. 76, pp.4350-4354; Gershoni and Palade, 1983, Analyt. Biochem., vol. 131, pp.1-15; Kuhajda, et al., 1989, Proc. Natl. Acad. Sci., USA, vol. 86, pp.1188-1192. In some instances blots were visualized with a dye-baseddetection system. Immunoblots were transferred and processed throughprimary antibody incubation. Biotinylated goat anti-rabbit IgG (VectorLaboratories) was used as a secondary antibody, followed withintervening washes by an avidin-horse radish peroxidase conjugate(Vector Laboratories) and developed with a Biomeda Universal Substratekit containing 3-amino, 9-ethyl carbazole (Biomeda Laboratories) usedaccording to the manufacturer's directions.

Cell line A₂₀ was obtained from the American Type Culture Collection(ATCC), Rockville, Md. All media were supplemented with 2 mML-glutamine, 100 u/ml penicillin, and 100 μg/ml streptomycin, andiron-supplemented newborn calf serum (Hyclone) at the indicatedconcentrations. All cells were grown in a humidified 5% CO₂ atmosphereat 37°. A₂₀ cells (Kim, et al., 1979, J. Immunol., vol. 129, pp.549-554) were grown in RPMI 1640 medium supplemented with 10% serum.

EXAMPLE 3

Peptide mapping of pp42, pp35 and pp32

The structural relationship between pp42, pp35 and pp32 was examinedusing high-resolution two dimensional peptide mapping. Peptide mappingwas performed using the Elder technique (Elder, et al., 1977, J. Biol.Chem., vol. 252, pp. 6510-6515; Speicher, et al., 1980, Proc. Natl.Acad. Sci., USA, vol. 77, pp. 5673-5677). FIG. 3 shows the peptide mapsof each species alone, and in combination with each of the otherspecies. Note in the top row that the general pattern of the maps issimilar, but that each map is unique. The combined maps show that whilesome peptides overlap, many are unique; in this system, the intensity ofnon-overlapping peptides is reduced relative to the individual maps,while overlapping peptides remain essentially unaltered. Importantly,one cannot simply derive the map of pp35 or pp32 from the map of pp42;likewise, the map of pp35 does not wholly contain the map of pp32. Thissuggests that the relationship between the proteins is more complex thanone whereby the two smaller polypeptides would be derived from pp42 byproteolytic cleavage.

EXAMPLE 4

Purification of pp35 and pp32

The initial purification scheme using cross-linked myosin failed toprecipitate pp32 and resulted in low yields of pp35. For these reasons,anti-pp35d was used to assay fractions during development of analternative purification strategy, consisting of sequential detergentlysis in low-ionic strength buffer, DEAE-cellulose chromatography, HPLCanion-exchange chromatography, and HPLC hydroxylapatite chromatography.The Coomassie-stained gel shown in FIG. 4a illustrates typicalsequential fractions from the stage of HPLC anion exchange; this stepfails to completely resolve pp35 and pp32 (panel A), and includes afaint 42 kilodalton band present in the middle and right-hand lanes. Thecoomassie-stained lanes shown in panels B and C indicate that subsequenthydroxylapatite chromatography achieves essentially complete purity ofgreater than about 98%. FIG. 4b, lane B shows that the faint 42 kDa bandseen in FIG. 4a is also immunoreactive with anti-pp35d. Generally,100-200 μg of each protein could be purified from approximately 5×10⁹cells, representing an estimated yield of around 20%.

In a typical preparation, 6 liters of A₂₀ cells were harvested bycentrifugation at 600×g, 4°, for 15 min. then washed three times withunsupplemented RPMI 1640. For lysis, the cell pellet was resuspendedwith a pipette in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100(Pierce Chemical Co.), 10 mM sodium pyrophosphate, 2 mM sodium vanadate,3 mM ATP, 50 mM NaF, 0.5 mM diisopropyl fluorophosphate (DFP), 0.1 mMphenyl-methylsulfonyl fluoride (PMSF) to a density of 2×10⁸ cells/ml.The lysate was then centrifuged at 17,500×g for 20 min. at 4°, and thesupernatant passed through a 0.2 μ filter before application to a 2.5×10cm DEAE cellulose column (DE53, Whatman) pre-equilibrated in 150 mMNaCl, 20 mM Tris-HCl, pH 7.5 containing 2 mM sodium vanadate, 0.5 mMDFP, 0.1 mM PMSF. The column was then eluted with a 400 ml lineargradient to 600 mM NaCl in the same buffer with the eluate collected in70 equal fractions. Fractions were electrophoresed on 10% Laemmli gels(Laemmli, 1970, Nature vol. 227, pp. 680-685) which were furtheranalyzed by Western blotting using anti-pp35d. Positive fractionscontaining pp42, pp35 and pp32 eluting around 400 mM naCl were pooledand applied directly to an HR-5/5 MonoQ FPLC column (pharmacia FineChemicals) pre-equilibrated in 150 mM NaCl, 20 mM Tris-HCL, pH 8.5, roomtemperature, 1 mM β-mercaptoethanol, 0.1 mM PMSF at a flow rate of 2ml/min. The column was then developed with a 60 ml gradient of 150 mMnaCl in the same buffer to 1.0M NaCl in the same buffer at pH 7.0. Each600 μl fraction was analyzed on Coomassie-stained Laemmli gels and byWestern blotting as previously described. Under these conditions, pp42,pp35 and pp32 eluted at approximately 0.78M NaCl. Following HPLC,fractions containing pp35 or pp32 were individually pooled and appliedseparately to a 30×4.6 mm MAPS HPHT analytical HPLC hydroxylapatitecolumn (Bio-Rad) after 1:1 dilution in 10 mM sodium phosphate, pH 6.8 10μM CaCl₂, 0.1 mM PMSF. The column was developed with an 18 ml lineargradient to 600 mM sodium phosphate, 10 μM CaCl₂, 0.1 mM PMSF at a flowrate of 0.3 ml/min. This purification protocol resulted in homogeneouspreparations of pp35 and pp32 on Coomassie-stained Laemmli gels.

Purified pp35 and pp32 were used to raise another set of polyclonalantibodies. Using material purified as described above. 100 μg of eitherpp35 or pp32 in 2 ml of complete Freunds adjuvant were injected intofour subcutaneous sites in each of two Pasteurella-free New ZealandWhite rabbits on Day 0. On Day 14, the rabbits were boosted with thesame amount of antigen in incomplete Freund's adjuvant, with bimonthlybleedings commencing on Day 28. Antibody production was monitored byWestern blotting. Initially, in IgG fraction was prepared by loadingserum aliquots onto a 5 ml protein A-Sepharose CL4B (Pharmacia) columneluted with 0.1M glycine pH 2.7 and collected into 0.5M sodium phosphatebuffer pH 7.5. Fractions were concentrated up to 25 mg/ml IgG by vacuumdialysis against phosphate buffered saline containing 0.1 mM PMSF, 1 mMβ-mercaptoethanol. These antibodies, designated anti-pp35n andanti-pp32n, were affinity-purified as described below and characterizedfor use in quantitative immunoblotting, immunoprecipitation, andimmunohistochemical localization studies.

For affinity-purification of anti-pp35n, an affinity column was preparedby concentrating the protein on an 0.4 ml hydroylapatite column(Fast-Flow, Calbiochem) to 600 μg/ml in the eluting 0.4M sodiumphosphate buffer, then reacting 600 μg of pp35 with 1 ml of EAHSepharose CL4B (Pharmacia) and 2 mg each of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl andN-hydroxysulfosuccinimide (Pierce Chemical Co.) for 6 h at roomtemperature under constant stirring. Any remaining reactive groups werecapped by incubation with 0.5M Tris HCl, pH 7.5, for 2 h. After washing,the derivatized resin was stored in phosphate-buffered saline containing12 mM sodium azide. A similar column was prepared using 200 μg of pp32coupled to 1.4 ml of resin. To affinity-purify the antibodies, 0.8 ml ofIgG solution were run into a column and incubated for 1 h at roomtemperature. The column was washed with 15 ml of 2×phosphate-bufferedsaline containing 0.05% Tween 20, and the antibody eluted with 3 ml of0.1M glycine pH 2.7. The antibody was collected into 1 ml of 0.5M sodiumphosphate, pH 7.5 and concentrated by vacuum dialysis. FIG. 5illustrates the specificity of these antibodies in Western blots of A₂₀cell lysates; lane A shows that anti-pp35n reacts primarily with pp35,while lane B shows that anti-pp32n reacts primarily with pp32. Thecross-reactivities are informative as well. Anti-pp35n shows minorcross-reaction with pp32, pp42, and an unidentified band, whileanti-pp32 cross-reacts with pp35. This pattern demonstrates that pp35and pp32 are unique proteins sharing some epitopes in common with eachother, and, in the case of pp35, with pp42. Moreover, theseimmunochemical data independently support the conclusions drawn from theanalysis of peptide maps.

EXAMPLE 5

Induction of pp35 and pp32 in resting B cells

The difference between the staining observed in normal tissues and inA₂₀ cells reflects differences in proliferative and functional state ofthe cell samples. Purified small, dense, resting B cells expressrelatively low levels of pp35 and pp32 until they are activated anddriven into proliferation by a well-characterized polyclonal B cellstimulator, bacterial lipopolysaccharide (See Rabin, et al., 1986, J.Exp. Med., vol. 164, pp. 517-531; Coffman, et al., 1986, J. ImmunoI.,vol. 136, pp. 4538-4541). The data, shown in FIG. 6a, demonstrate thatover the course of 72 hours, the expression of pp35 rose 12-fold from5.9×10⁴ to 7.1×10⁵ copies per cell (panel C), while pp32 increased7.4-fold from 7.0×10⁴ to 5.2×10⁵ per cell (panel D). Because cellstimulation involves a global increase in cell size, these data werealso normalized to total cellular protein so as to reflect specificinduction over and above the general increase. Shown in Panels A and B,these data demonstrate a 3.8-fold specific induction of pp35, and a2.3-fold induction of pp32. Preliminary studies measuring tritiatedthymidine incorporation by resting B cell cultures as a function of timeand LPS concentration (data not shown) yielded stimulation patterns over72 h consistent with those reported in the literature. Expression ofpp35 and pp32 was quantified by ¹²⁵ I-protein A immunoblotting inconjunction with computerized image analysis, which yielded anintegrated optical density for each band on an autoradiograph;integrated optical density values were converted to protein measurementsthrough a parallel calibration curved of purified pp35 and pp32. FIG. 6billustrates the immunoblots from which FIG. 6a was derived.

Six to eight week old BALB/C mice were obtained from Charles river.Small dense B cells were isolated from spleen on Percoll gradientsessentially as described (Rabin, et al., 1986, J. Exp. Med., vol. 164,pp. 517-531; Coffman, et al., 1986, J. Immunol., vol. 136, pp.4538-4541). T cells were removed using a cocktail of hybridoma culturesupernatants including C3PO (Vidovic, et al., 1984, J. Immunol. vol.132, pp. 1113-1117), an anti-Ly-1, JIJ (Bruce, et al., 1981, J.Immunol., vol. 127, pp. 2496-2501), an antiThy 1.2, RL172 (Ahmed, etal., 1988, J. Virol., vol. 62, pp. 2102-2106), an anti-CD4, and 3155(Sarmiento, et al., 1980, J. Immunol., vol. 125, pp. 2665-2672), ananti-CD8, together with 10% Low-Tox-M rabbit complement (CedarlaneLaboratories). The T cell reagents were the gracious gift of Dr. DrewPardoll. To measure activation by lipopolysaccharide, cells at 1.5×10⁵cells per well were plated into 96 well tissue culture plates in a totalvolume of 220 μl per well. Cells were activated by incubation at 37° for48 h with increasing doses of lipopolysaccharide (LPS W E.coli 0127:B-8,Difco Laboratories). The cells were then pulsed with 1 μCi/well of ³ Hthymidine (ICN) for 16 h, then harvested with a cell-harvester ontoWhatman glass-fiber filters presoaked in 10 mM thymidine. This resultedin a dose-dependent stimulation of thymidine incorporation of up to140-fold over a range of 0.08-50 μg/ml LPS. Cell viability wasdetermined by amido schwarz dye exclusion, and was 95% for stimulated Bcells at 72 h, and 50% for unstimulated cells.

To determine pp35 and pp32 content by quantitative Western blotting,resting B cells were incubated at the above concentration in T-150culture flasks for 0 h, 1 h, 24 h, 48 h and 72 h in the presence of 40μg/ml LPS. At each time point, cells were counted harvested, lysed byresuspension in 150 μl of 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% TritonX-100, 0.1 mM PMSF, 0.5 mM DFP. The supernatants were then collected bycentrifugation at 16,000×g for 10 min. at 4° and stored at -80°;separate aliquots wee reserved for determination of total protein by BCAprotein assay (Pierce Chemical Co.). Similar duplicate amounts of totalcellular protein for each time point were separated on 10% Laemmli gelsalong with a standard curve prepared from a mixture of purified pp35 andpp32 calibrated by BCA protein assay. The standard curve consisted of 31ng, 62.5 ng, 125, ng, 250 ng, and 500 ng each of pp35 and pp32. The gelswere transferred to nitrocellulose and the blots probed in the samesolution of a mixture of affinity purified anti-pp35n and anti-pp32n at5 μg/ml in Tris-saline containing 3% bovine serum albumin for 2 hfollowed, after intervening washes, by ¹²⁵ I protein A at 3×10⁶ cpm/mlin albumin-Tris saline for 2 h. The blots were washed togetherextensively in Tris-saline, and exposed on the same piece of Kodak XAR-5film in a cassette containing a Cronex Lightning Plus intensifyingscreen (DuPont). The bands seen on the autoradiogram were quantitated bycomputed densitometry using a Loats image analysis system. Theintegrated optical density values obtained for the standard values ofpp35 and pp32 were plotted against he amount of protein.

EXAMPLE 6

pp35 and pp32 expression in lymphoid cell lines

In populations of neoplastic cell lines, pp35 and pp32 are expressed athigh levels (FIG. 7). In the majority of cell lines, these levels exceedthose seen in normal B cells stimulated with LPS for 72 h. To a firstapproximation, there is a reciprocal relationship between expression ofpp35 and pp32 in that less differentiated lines tend to express higherlevels of pp35 than pp32, while for more differentiated lines thereverse is true. There are at least two possible explanations for thediscordant case of the cell line BCL₁ : pp32 expression may be low; orpp32 itself may be immunologically altered. While these data are open toa number of interpretations, one explanation is that pp35 and pp32assume different functions in the cell nucleus which are linked todifferent states of proliferation or differentiation.

2PK-3, a BALB/C B cell lymphoma (Lanier, et al., 1981, J. Immunol., vol.127, pp. 1691-1697), and ABE-8.1/2, a BALB/C pre-B cell lymphoma(Burchiel and Warner, 1980, J. Immunol., vol. 124, pp. 1016-1021), weregrown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5g/l glucose, 50 μM 2-mercaptoethanol, and 10% serum. P815, a DBA/2mastocytoma (Ralph, et at., 1976, J. Exp. Med., vol. 143, pp.1528-1533), and P3.6.2.8.1, a subline of the BALB/C plasmacytoma MOPC 21(Knopf, et al., 1973, Eur. J. Immunol., vol. 3, pp. 251-259), were grownin DMEM with 10% serum. BCL₁ Clone CW13.30-383, a BALB/C B cell leukemia(Brooks, et al., 1984, J. Immunol., vol. 133, pp. 3133-3137), was grownin RPMI 1640 with 10% serum.

EXAMPLE 7

pp35 and pp32 are phosphoproteins

The initial evidence that pp35 and pp32 are phosphoproteins came fromsimultaneous autoradiography and immunoblotting of these proteins afterpurification from cells labeled with ³² P as orthophosphate. In oneexperiment, A₂₀ cells were labeled for 4 h in otherwise phosphate-freemedium prior to purification. For each of the pp35 and pp32 panels ofFIG. 8, lane A illustrates a colorimetric immunoblot developed witheither anti-pp35n or anti-pp32n, and lane B represents an autoradiographof the blot. Both pp35 and pp32 are phosphoproteins in vivo. In thisexperiment, complete coincidence of the immunoreactivity of each proteinwith the radioactive band was observed. Moreover, ³² P-labeled pp35 andpp32 are radiochemically pure, so that measurements of radiochemicalactivity truly reflect incorporation into each protein.

EXAMPLE 8

Casein kinase II phosphorylates pp35 and pp32

The potential role of various kinases in pp35 and pp32 phosphorylationwas studied in vitro by their ability to phosphorylate native anddephosphorylated pp35 or pp32. Labeled pp35 contains phosphoserine andphosphothreonine, while labeled pp32 contains only phosphoserine;alkaline phosphatase quantitatively dephosphorylates ³² P-phosphoserinein both pp35 and pp32.

A₂₀ cells were labeled in vivo. Approximately 5×10⁹ A20 cells werewashed once and resuspended at 2×10⁷ cells/ml in phosphate-free minimalessential medium (Gibco) supplemented with 10% iron-supplemented calfserum previously dialyzed against medium, 2 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin, and 1× non-essential amino acidssolution. The cells were incubated in humidified 5% CO₂ atmosphere at37° with 15 mCi of carrier-free ³² P as orthophosphate (Amersham) for 3h. Following incubation, pp35 and pp32 were purified as described above.

Studies of several kinase in vitro showed that both pp35 and pp32 aresubstrates for casein kinase II. The results strongly suggest aphysiologic role for casein kinase II in pp35 phosphorylation, and for arelated kinase in pp32 phosphorylation. Casein kinase II consistentlyyielded the highest degree of ³² P incorporation onto dephosphorylatedpp35 and pp32, rephosphorylating pp35 to a level of 0.76 mol/mol (TableI).

                  TABLE I                                                         ______________________________________                                        Casein Kinase II                                                                          pp35, dephosphorylated                                                                        0.76 mol/mol + 0.03                                           pp35, native    0.05 mol/mol + 0.01                                           pp32, dephosphorylated                                                                        0.08 mol/mol + 0.01                                           pp32, native   <0.01 mol/mol                                      Protein Kinase A                                                                          pp35, dephosphorylated                                                                       <0.02 mol/mol                                                  pp35, native   <0.02 mol/mol                                                  pp32, dephosphorylated                                                                       <0.02 mol/mol                                                  pp32, native   <0.02 mol/mol                                      Protein Kinase A                                                                          pp35, dephosphorylated                                                                       <0.01 mol/mol                                                  pp35, native   <0.01 mol/mol                                                  pp32, dephosphorylated                                                                       <0.01 mol/mol                                                  pp32, native   <0.01 mol/mol                                      EGF Receptor Kinase                                                                       pp32, dephosphylated                                                                         no incorporation                                               pp32, dephosphorylated                                                                       <0.01 mol/mol                                      ______________________________________                                    

In contrast, the catalytic subunit of protein kinase A, protein kinaseC, and EGF receptor kinase all failed to promote significant levels ofincorporation, and showed little sensitivity to whether the substrateproteins were dephosphorylated or not. As would be predicted for caseinkinase II activity, phosphorylation of both pp35 and pp32 by caseinkinase II utilized GTP as a substrate and was completely inhibited byheparin.

Rephosphorylation of the dephosphorylated phosphate turnover sites wasinvestigated with protein kinase C from rat-brain, the catalytic subunitof cAMP-dependent protein kinase (Sigma), casein kinase II from bovinethymus, and A 431 cell epidermal growth factor receptor kinase,generously provided as a solution of 30 μg/ml of EGF receptor in buffercontaining 50 μM EGF by Dr. Wolfgang Weber (Instit fur physiologischeChemie, Universitat Hamburg).

Casein kinase II was purified as described (Zandomeni et al., 1988, FEBSLetters, vol. 235, pp. 247-251). Aliquots of fractions were screened forcasein kinase II activity in 30 μl final volumes of 20 mM Tris HCl at pH7.5 160 mM NaCl, 8 mM MgCl₂, 0.1 mM DTT, 0.1 mM GTP with gamma-labeled³² P GTP (Amersham) at a final specific activity of 1 μCi per nanomole,1.0 mg/ml partially hydrolyzed and dephosphorylated casein (Sigma), inthe presence and absence of the casein kinase II inhibitor5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB, Sigma) at aconcentration of 100 μM, added from a stock solution in 50% dimethylsulfoxide. After incubation at 30° for 10 min, reactions were terminatedby the addition of 500 μl of ice-cold 25% TCA. Fractions showing kinaseactivity 50% inhibitable by DRB were selected for pooling. The finalenzyme preparation showed the expected subunit structure onCoomassie-stained Laemmli gels, utilized GTP, showed partial inhibitionby DRB, and showed complete inhibition by 1 μg/ml heparin.

Dephosphorylation of pp35 and pp32 was carried out by incubation with 1unit of alkaline phosphatase from bovine intestinal mucosa (Sigma, TypeVII) per μg of protein in 20 mM Tris HCl at pH 8.0, 1 mM MgCl₂, 0.1 mMZnCl₂ for 1 h at room temperature. The reaction mixture was loaded ontoa 0.2 ml hydroxylapatite column (high resolution, Calbiochem),previously equilibrated in 20 mM sodium phosphate pH 7.5. The column waseluted sequentially with 0.4 ml each of 100 mM, 200 mM and 450 mM sodiumphosphate, pH 7.5. Alkaline phosphatase eluted at 100 mM sodiumphosphate, while pp35 or pp32 eluted at 450 mM. Proteins labeled in vivowith 32p as orthophosphate were used to estimate the extent ofdephosphorylation. While the ³² P label was quantitatively removed frompp32, pp35 retained approximately one-third of the original label in theform of phosphothreonine.

Protein kinase C was purified using elements of two protocols (Walton etal., 1987, Analyt. Biochem., vol. 161, pp. 425-437; Woodgett and Hunter,1987, J. Biol. Chem., vol. 262, pp. 4836-4843). Briefly, eleven ratbrains were harvested, immediately frozen in liquid nitrogen, andhomogenized with a Brinkmann Polytron in 150 ml of 10 mM Tris HCl at pH7.5, 10 mM EGTA, 5 mM EDTA, 0.1% (v/v) β-mercaptoethanol, 4 μg/mlleupeptin, 0.5 mM diisopropylfluorophosphate, 0.1 mM phenylmethylsulfonyl fluoride, and 6 μg/ml soybean trypsin inhibitor. The homogenatewas centrifuged for 30 min at 10,000×g. The supernatant was filteredthrough three layers of cheesecloth into DE53 (Whatman) pre-equilibratedin 20 mM Tris HCl at pH 7.5, 1 mM EDTA, 0.1% (v/v) β-mercaptoethanol andbatch adsorbed for 30 min while stirring at 4°. The resin was pouredinto a 2×20 cm column which was then eluted with a 1 1 gradient of 20 mMNaCl to 300 mM NaCl in the same buffer. The column fractions werescreened for phorbol ester stimulated activity by incubating 5 μl of 1mg/ml histone H-1 (Boehringer-Mannheim) in a final volume of 20 μlcontaining 20 mM HEPES, pH 7.5, 10 mM MgCl₂, 0.5 mM CaCl₂, 50 μg/mlfreshly sonicated phosphatidylserine, 0.1 mM ATP with gamma-labeled ³²P-ATP (Amersham) at a final specific activity of 1 μCi per nanomole,and, in some samples, phorbol myristic acetate at 5 μg/ml. Afterinitiation by the addition of substrate stock, the reactions werestopped after 5 min at 30° by the addition of 500 μl of 20% ice cold 20%trichloroacetic acid (TCA). Precipitates were incubated at 4° for 20min, then centrifuged for 5 min at 16,000×g. The pellets were washedtwice with 20% TCA and then counted by Cerenkov counting. Positivefractions were selected on the basis of PMA-stimulated phosphorylationof histone H-1, pooled, and then brought to 1.5M NaCl by the addition ofsolid NaCl. This pool was then loaded onto a 10 ml phenyl Sepharosecolumn (Pharmacia) pre-equilibrated in 20 mM Tris HCl at pH 7.5, 2 mMEDTA, 2 mM EGTA, 1 mM dithiothreitol, 1.5M NaCl. The column was elutedwith a 200 ml linear gradient of 1.5M NaCl to 0M NaCl in the samebuffer. Positive fractions were pooled and applied to a 10 mlprotamine-agarose column (Pharmacia) equilibrated in 20 mM Tris HCl atpH 7.5, 2 mM EGTA, 2 mM EDTA 1 mM DTT 0.1 mM PMSF, 0.5 mM DEP 0.1M NaCLpH 7.5. The column was eluted with a 160 ml linear gradient ofincreasing salt from 0.5M to 1.5M NaCl. Purified protein kinase Cactivity comigrated with an 80 kDa band on Coomassie-stained Laemmligels; PMA increased histone H-1 phosphorylation by 15 to 18 fold overbaseline samples without PMA in typical preparations. The final yieldwas approximately 40 μg. The enzyme was stored stably for several monthsat -80° in buffer containing 16% glycerol and 0.02% Triton X-100.

Protein kinase C assays were carried out for 30 min at 37° with 0.5 μgof the 80 kDa protein kinase C isoenzyme in 20 mM HEPES at pH 7.5, 10 mMMgCl₂, 0.5 mM CaCl₂, 50 μg/ml freshly sonicated phosphatidylserine, and0.1 mM containing gamma-labeled ³² P ATP (Amersham) at a final specificactivity of 5 μCi per nanomole, in the presence and absence of 5 μg/mlPMA. Generally, 1 to 5 μg of pp35 or pp32 were tested. Reaction productswere analyzed on Laemmli gels and on two dimensional gels (O'Farrell,1979, J. Biol. Chem., vol. 250, pp. 4007-4021) gels using historic H1(Boehringer) as a positive control substrate.

Protein kinase A assays utilized the catalytic subunit of cAMP-dependentprotein kinase from bovine heart (Sigma). Reactions were carried out byincubating 50 units of enzyme with 5 μg of either pp35 or pp32 forvarying times at 30° in 120 μl volumes of 50 mM HEPES at pH 7.4, 25 mMNaCl, 10 mM MgCl₂, 1 mM EDTA, 0.1 mM β-mercaptoethanol, and 0.1 mM ATPcontaining gamma-labeled ³² P ATP at a final specific activity of 1 μCiper nanomole. 1 μg histone H1 served as a positive control substrate.Reaction products were analyzed as above.

Casein-Kinase II assays were carried out by incubating 1 μg enzyme with1 to 5 μg pp35 or pp32 for varying times at 30° in 20 mM Tris HCl at pH7.9, 8 mM MgCl₂, 0.1 mM DTT and 0.1 mM GTP containing gamma-labeled ³² PGTP at a final specific activity of 1 μCi per nanomole. Partiallydephosphorylated and hydrolyzed casein from bovine milk (Sigma) servedas positive control substrate. 1 μg/ml of heparin in the reactionmixture completely inhibited phosphate incorporation into casein, pp35,and pp32. Reaction products were analyzed as above.

The epidermal growth factor receptor assays were carried out byincubating 0.15 μg kinase with 1 to 5 μg pp35 or pp32 for varying timesat 30° in 100 μl of 20 mM HEPES at pH 7.5, 1 mM MnCl₂, 5 mM MgCl₂, 50 mMEGF, and 0.1 mM ATP containing gamma-labeled ATP at a final specificactivity of 1 μCi per nanomole. The autophosphorylation of the receptorserved as a positive control for the kinase activity. Reaction productswere analyzed as above.

The amount of phosphate transferred from ATP or GTP to pp35 or pp32 wascalculated from Cerenkov counts of the corresponding excised bands fromCoomassie-stained gels. Raw Cerenkov counts were converted to moles ofphosphate using an experimental specific activity value of Cerenkovcounts per minute per nMol phosphate obtained from triplicate counts of5 μl aliquots of reaction mixtures as known nucleotide concentration;care was taken to closely approximate the sample geometry used tomeasure the activity of the gel slices. These measurements were combinedwith previous triplicate measurements of protein substrate concentrationby the BCA assay (Pierce Chemical Co.) to calculate the stoichiometry ofphosphorylation at the susceptible sites. The background countssubtracted from each measurement were obtained by counting gel slices ofcomparable area from each lane from areas with no protein. Kinasereactions were carried out in triplicate for varying time points up to 2h to assure completion. Casein kinase II reactions were complete by 15min, while protein kinase A, protein kinase C, and the EGF receptorkinase all showed small, gradual increases over the two hour period withno obvious plateau. Two hours were therefore arbitrarily chosen as acutoff for the determination of phosphorylation stoichiometry for theselatter kinases.

EXAMPLE 9

HeLa cells contain pp35 and pp32

HeLa cells, a human cervical epithelioid carcinoma cell line, (availablefrom the ATCC) appear to contain pp35 and pp32. An immunoblot of a totalHeLa cell hypotonic detergent lysate was prepared essentially asdescribed for A₂₀ cells above. Antibodies to denatured, gel-purifiedpp35 which recognize both pp35 and pp32 react with a diffuse band atapproximately 33 kDa; in lighter exposures the heavy band resolves intotwo closely-spaced components. The experiment clearly indicates thepresence of cross-reactive species of the expected molecular weights ina human cell line, but does not clearly establish the number of speciesor degree of relationship with murine lymphoid pp35 or pp32.

EXAMPLE 10

pp35 and pp32 immunostaining correlates with increased malignantpotential

Human tissues react with antibodies to native murine pp35 and pp32,showing increased staining frequency, increased staining intensity, andaltered subcellular distribution with increasing malignancy.

Paraffin-embedded sections of human lymphoid tissue were stained withaffinity-purified antibodies to native pp35 and pp32. Staining wasevaluated semiquantitatively by two independent observers. In a diffuselarge cell lymphoma, the neoplastic cells show prominent nuclearstaining while the normal lymphocytes are negative. Adenomatous colonicepithelium shows prominent nuclear anti-pp35 staining in virtually everycell, in contrast to the limited staining of crypts seen in normalcolon. In contrast, invasive adenocarcinoma of the colon showspredominantly cytoplasmic pp35 staining, while pp32 staining remainsnuclear. This change from nuclear to cytoplasmic distribution is highlyreminiscent of the association of c-abl transforming activity with arelocation from nucleus to cytoplasm (Van Etten, et al., Cell, vol. 58,pp. 669-678, 1989).

Table 2 illustrates the results of a screen of human lymphomas withanti-pp35 and anti-pp32, which demonstrates that increased stainingfrequency and intensity and altered distribution are associated withincreased malignancy. Lymphomas were examined for pp35 and pp32 stainingindependent of diagnosis, then ranked in increasing order of frequencyof staining. The result was a ranking which predicted the level ofvirulence suggested by the diagnosis.

                                      TABLE 2                                     __________________________________________________________________________    pp35 and pp32 Staining in Normal and Neoplastic Lymphoid Tissue                               pp35          pp32                                                            %             %                                               LESIONS         Positive                                                                          Intensity                                                                          Location                                                                           Positive                                                                          Intensity                                                                          Location                               __________________________________________________________________________    LOW GRADE LESIONS                                                             Small           40-50%                                                                            +1   Nucleus                                                                            NOT DONE                                        Lymphocytic     Staining in tumor only. Weak,                                 Malignant       infrequent staining of normal                                 Lymphoma        small lymphocytes                                             (Well-                                                                        Differentiated                                                                Lymphocytic)                                                                  Follicular      40-50%                                                                            +/-  Nucleus                                                                            40-50%                                                                            +1   Nucleus                                Lymphoma Small                                                                Cleaved                                                                       Predominant                                                                   (Poorly                                                                       Differentiated                                                                Lymphocytic                                                                   Lymphoma)                                                                     INTERMEDIATE GRADE LESION                                                     Follicular      60-70%                                                                            2+   Cytoplasm                                                                          60-70%                                                                            +3   Nucleus                                Lymphoma Large      1+   Nucleus                                              Cell Predominant                                                                              Staining described is for large                                                             Small cells showed 40-50% +1                    (Nodular        cell component. Small cells                                                                 nuclear staining.                               Histiocytic     showed +/- nuclear staining in                                Lymphoma)       40-50% of cells.                                              HIGH GRADE LESIONS                                                            Diffuse-Large   >90%                                                                              3+   Nucleus                                                                            >90%                                                                              +3   Nucleus                                Cell Malignant  Focal                                                                             2+   Cytoplasm                                            Lymphoma        Small, normal lymphocytes                                                                   Small, normal lymphocytes                       (Diffuse        negative      negative                                        Histiocytic                                                                   Lymphoma)                                                                     Small           >90%                                                                              3+   Nucleus                                                                            >90 3+   Nucleus                                Noncleaved Cell Small, normal lymphocytes                                                                   Small, normal lymphocytes                       Malignant       negative      negative                                        Lymphoma                                                                      (Diffuse                                                                      Undifferentiated                                                              Lymphoma)                                                                     NORMAL TISSUES                                                                Tonsil &        50-60%                                                                            1+   Nucleus                                                                            50-60%                                                                            1+   Nucleus                                Reactive Lymph           Germinal      Germinal                               Node (Identical          Centers       Centers                                Findings)       30-60%                                                                            1+   Nucleus                                                                            30-40%                                                                            1+   Nucleus                                                         Paracortex    Paracortex                                             Occasional small faci of 3+ with                                                            Occasional small foci of 3+ with                                70% positive nuclear staining in                                                            70% positive nuclear staining in                                paracortex.   paracortex.                                     __________________________________________________________________________

EXAMPLE 11

pp32 cDNA is cloned and partially sequenced

Screening an oligo dT-primed A₂₀ λ gt11 cDNA library with a 42 baseoligonucleotide guessmer (Sambrook, et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp.11.11-11.16) derived from pp35 peptide sequence recovered a pp35-relatedcDNA predicting additional independent pp35 peptide sequence. The majorpp35 phosphopeptide sequence L-L-P-Q-L-S-Y-L-D-G-Y-D-D-E (SEQ ID NO: 6)containing a casein kinase II phosphorylation, site was backtranslatedto the best guess 42 base oligonucleotide sequence using previouslydescribed codon preference rules (Lathe, J. Mol. Biol., vol. 183, pp.1-12, 1985). The resultant oligonucleotide,5'-CTGCTGCCCCAGCTGTCCTACCTGGATGGCTATGATGATGAG (SEQ ID NO: 8), hybridizeswith a 1.3 kb RNA species in Northern blots of A₂₀ cell RNA.

Approximately 600,000λgt11 plaques were screened with end-labeledoligonucleotide and the filters washed at moderate stringency, yielding23 cDNA clones which remained positive through tertiary plaquepurification. The first cDNA to be subcloned into Bluescript™ andpartially sequenced by double-stranded dideoxynucleotide sequencing isapproximately 1 kb and contains predicted protein sequences identical toan independently sequenced pp35 peptide. A portion of the sequence ofthe cDNA is shown in FIG. 9.

The cDNA (SEQ ID NO: 4) and predicted peptide sequences (SEQ ID NO: 5)were compared to all nucleotide and protein sequences in the Genbank andEMBL libraries using the FASTA program of the University of Wisconsin inGCG sequence analysis package, and using the TFASTA program to comparepredicted amino acid sequences to translations of the nucleotidesequences. No close matches were found, confirming that pp32 is distinctfrom previously described nuclear proteins. pp32 cDNA does containinteresting homologies of 30 to 50% over 60 to 100 nucleotide stretcheswith such molecules as human calcyclin, human c-myc germ lineprotooncogene, human calmodulin, and human U2 snRNP, however the trueextent and significance of these homologies is at present unknown. Nohomology has been found in the determined sequence to erythrocyteprotein 4.1, even though some preparations of anti-4.1 antibodiescross-react with denatured pp35. Curiously, there is no suchcross-reactivity with native pp35. This case is reminiscent of synapsinI, which also cross-reacts with anti-protein 4.1 antibodies but isunrelated at the sequence level (Baines and Bennett, (1985), Nature,vol. 315, pp. 410-413; McCaffery and DeGennaro (1986), EMBO J., vol. 5,pp. 3167-3173; and Conboy, et al., (1986), Proc. Natl. Acad. Sci. USA,vol. 83, pp. 9512-9516.)

EXAMPLE 12

Cloning of Murine and Human pp32

The cloning strategy employed a 42 base pair non-degenerateoligonucleotide probe back-translated from an independently sequencedmurine pp35 peptide to screen a murine cDNA library from A₂₀ cells. Theinitial murine clones were used to obtain human cDNA and to complete themurine sequence.

Materials

A poly-T primed cDNA library was constructed by Clonetech from A₂₀ cellmRNA purified by the guanidinium isothiocyanate method (8) in lambdagt11. A randomly primed cDNA library from HL-60 cells in Lambda-Zap anda poly-T primed A₂₀ cell cDNA library in Uni-Zap were purchased fromStratagene. All cDNA's were subcloned into Stratagene pBluescript II KS+except for clones in vivo excised from the HL-60 Lambda-Zap librarywhich were in pBluescript I SK-. A GAPDH probe was purchased fromClonetech. Oligonucleotides were synthesized by Genosys. Enzymes andkits for molecular biology were from Amersham, Collaborative Research,Boehinger Mannheim, Clonetech, Gibco BRL, Stratagene, Promega,Schleicher and Schuell, and United States Biochemicals. Other chemicalspurchased from Sigma, and J. T. Baker. Radiochemicals were from Amershamand ICN.

Cell Culture

Cell culture media and reagents were from Gibco BRL, and serumsupplements were from Hyclone. A₂₀ cells (2, American Type CultureCollection) were maintained in RPMI 1640 supplemented 10% fetal bovineserum, 50 units/ml penicillin and 50 μg/ml streptomycin and passed twiceweekly by diluting 1:10 in fresh medium.

HL-60 cells (11) were maintained in RPMI 1640 supplemented 10% fetalbovine serum, 50 units/ml penicillin and 50 μg/ml streptomycin andpassed twice weekly by diluting 1:10 in fresh medium. Primary rat embryofibroblasts were either purchased through BioWhittaker or weregenerously donated by Dr. Chi Dang. Cells were grown in low glucose DMEMcontaining 10% FBS, 50 units/ml penicillin and 50 μg/ml streptomycin.All cells were grown in humidified incubators at 37°, 5% CO2.

Purification of Total and Messenger RNA

For the A₂₀ library, total RNA was isolated from cultured A₂₀ cellsusing the guanidinium/cesium chloride method (8). Poly-A+ mRNA waspurified using an oligo (dT)-cellulose column (8). Total RNA and mRNAfrom HL-60 cells were isolated using a modified protocol of thesingle-step acid-phenol extraction (12) and the MiniRiboSep kit fromCollaborative Biomedical, respectively.

Library Screening

Initial clones were obtained by screening the A₂₀ library with a long,non-degenerate oligonucleotide probe designed by back-translating, usingLathe's codon usage frequencies (13), the phosphopeptide from murinepp35 sequenced in Example 11. The oligonucleotide was synthesized byGenosys and purified by HPLC. 600,000 pfu of the previously describedA₂₀ lambda gt11 library was initially plated on E. coli strain Y1090,transferred onto Nytran filters (Schleicher and Schuell) and fixed bybaking for 2 hours in an 80° vacuum oven. The screening oligonucleotideguessmer was end-labelled using T4 polynucleotide kinase (New EnglandBiolabs) and used to probe the library at moderate stringency. Filterswere hybridized overnight in 6×SSC, 1×Denhardt's, 0.05% sodiumpyrophosphate, and 100 μg/ml salmon sperm DNA at 42°, then washed 2times at room temperature in 6×SSC and 2 times at 50° in 6×SSC.

13 clones positive on duplicate filters were plaque purified and the DNAisolated from liquid lysates on cesium chloride gradients (8). cDNAInserts were cut out using EcoRI and ligated into pBluescript II KS+(Stratagene). Cesium-banded plasmid was sequenced using T7 and T3primers (Stratagene) and Sequenase II (United States Biochemicals) andnested-deletion constructs were made using the Exo/Mung deletion kit(Stratagene).

The initial partial murine pp32 clone isolated by this strategy was usedas a probe to identify the complete human homolog from Stratagene'sHL-60 randomly-primed library. 500,000 pfu's were plated using XL1-Bluecells, transferred onto Nytran filters, and UV-crosslinked withStratagene's Stratalinker. The partial murine insert was used as a probeand labelled by random-priming using Prime-It (Stratagene) andhybridized with the filters in 50% formamide, 5× SSPE, 0.1% SDS, and 100μg/ml salmon sperm DNA at 42°. Filters were washed 2 times at roomtemperature in 2×SSC, and 2 times at 55° in 0.2×SSC. Plaque purifiedclones were in vivo excised using XL1-Blue cells and R408 helper phage(Stratagene). Sequencing was performed as described above. Finally, thecomplete murine clone A202 was isolated by using a 5' HindIII fragmentfrom the human clone as a probe to screen the Stratagene A₂₀ cDNAlibrary.

FIG. 10A shows the sequence of human pp32 cDNA cloned from HL-60 cells.Amino acids differing in the murine sequence are shown immediatelyunderneath the human sequence. Amino acids missing from the murinesequence are shown as asterisks. Underlined peptides correspond topeptides independently sequenced from murine pp35 which are alsoapparently found in pp32. Doubly underlined residues shown in boldconstitute a possible leucine zipper; note that the candidate zipper isbounded by additional leucines in the sixth positions immediatelyupstream and downstream. Also note candidate nuclear localizationsequences spanning amino acids 60-68 and 108-116.

FIG. 10B diagrams the cDNA clones used to generate the data shown inFIG. 1B. Clones with an "HL" prefix are derived from the human HL-60cDNA library in lambda-ZAP. Clone m35.7 is from the murine A20 lambdagt11 cDNA library and clone mA202.1 is from the murine A20 cDNA libraryin Uni-Zap. The HL2 clone contains the most 5' sequence obtained,therefore all other clones are numbered with respect to its first base.The numbers on the right signify the total length of each clone. `AAA`designates clones containing poly-A tails. The EcoRI site in HL13 wasused to generate a DNA fragment from its extended 3' region. Similarly,the HindIII site in HL2 was used to generate a DNA fragment from 5' end.Both fragments were used as probes in Northern blotting.

EXAMPLE 13

cDNA Encodes pp32

The evidence that the cDNA's thus obtained encode pp32 is several-fold:1! the polypeptide expressed from a partial murine cDNA clone reactspreferentially with antibodies to native murine pp32; 2! antibodies tothe expressed murine pp32 fragment react specifically with pp32 inWestern blot analysis of total cellular lysates of A₂₀ cells (5); 3!recombinant human pp32 expressed in baculovirus reacts with antibodiesto murine pp32 and pp35; and 4! the in vitro translation product ofhuman pp32 cDNA co-migrates with pp32 and not pp35 in Laemmli gels. Asadditional supporting evidence, the murine pp32 sequence encodes threepeptides independently sequenced from purified murine pp35 which areapparently common to both proteins.

The immunologic identification of the murine and human cDNA clones bearsfurther discussion.

Western Blots

All protein samples were run on 10% SDS-PAGE system (14). They weretransferred to nitrocellulose filters (Schleicher and Schuell) instandard Tris/glycine/20% methanol transfer buffer and blocked overnightin 3% BSA/PBS. Primary antibodies were hybridized onto blots at roomtemperature in 1×tris-buffered saline containing 0.1% Tween 20 (TBS-T)for 1 hour with continuous shaking. After washing the blot at roomtemperature in TBS-T, a donkey anti-rabbit HRP secondary antibody wasapplied. Amersham's ECL chemiluminescent Western detection kit was usedto visualize binding of the antibodies.

FIG. 11 used an affinity-purified antibody to native pp32 designatedanti-pp32n (see Example 4), to analyze the expressed product of apartial murine clone corresponding to amino acids 67 through 247. FIG.11 shows a 30 second exposure of a Western blot probed with anti-pp32n.Lane A: 5 μg purified recombinant partial murine pp32 from clone 35.7;lane B: A₂₀ lysate.

Anti-pp32n specifically identifies pp32 in total cellular lysates of A₂₀cells (lane B) and reacts with the purified recombinant murine fragment(lane A). Anti-pp32n also reacted with the recombinant murine fragmentin lysates of transfected bacteria, but failed to react with proteins inuntransfected bacterial hosts.

Protein Purification and Analysis

Murine pp32 and pp35 were purified from A₂₀ cells as describedpreviously (see Example 4). A recombinant partial murine pp32 fragmentwas expressed and purified from CAG456 E. coli cells using the pRXexpression vector (10) containing cDNA encoding amino acids 67 through247 of murine pp32 (pRX32). A single colony of CAG456 containing thepRX32 was grown in 1 ml LB media with 50 μg/ml ampicillin at 30° withvigorous shaking. The saturated culture was then transferred into 50 mlLB with ampicillin and allowed to reach saturation overnight. Thesaturated culture was then added to 500 ml M9CA media (8) withampicillin and allowed to grow with vigorous shaking at 30° for 3.5 h.Indol acrylic acid was added to the cells to achieve 10 μg/ml finalconcentration, and growth was continued for another 4.5 h.

The cells were then pelleted at 4000×g at 4°, washed with 100 ml of 10mM Tris-HCl, pH 8.0 at 4°, pelleted again and lysed in ice-cold 140 mlSTET lysis buffer consisting of 8% sucrose, 10 mM Tris-HCl pH 8.0 at 4°,50 mM EDTA, 0.5% Triton X-100, 0.5 mM diisopropylfluorophosphate, and 10μg/ml each of chymostatin, leupeptin, antipain, and pepstatin. The celllysate was kept on ice and sonicated 10-11 times with 30 s bursts atintensity level 6 at 15 s intervals using a Brinkmann Sonificator. Celldebris was removed by centrifugation at 13,000×g for 10 min at 4°, thesupernatant was brought to 25% ammonium sulfate, and the lysate wasallowed to precipitate overnight on ice. The precipitate was pelleted at13,000×g for 10 min at 4°; the supernatant was then brought to 65%ammonium sulfate and allowed to further precipitate. The proteinprecipitate was pelleted as before, resuspended in 20 ml of 200 mM NaCl,20 mM Tris-HCl pH 7.5 at 4°, 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.1 mMphenylmethylsulfonyl fluoride and dialyzed overnight in the same bufferto remove ammonium sulfate.

The dialyzed lysate was applied to a HR-5/5 MonoQ FPLC column(Pharmacia) at a flow rate of 1 ml/min. The column was washed anddeveloped with a 50 ml linear gradient of 200 mM to 1.0 mM NaCl in thesame buffer and 1 ml fractions were collected. Samples were analyzed bySDS-PAGE (14) and Western blots probed with anti-pp32 polyclonalantibodies. Pure partial recombinant murine pp32 was usually obtainedafter only one column run. Material was rechromatographed under the sameconditions as needed to obtain >95% purity by SDS-PAGE. Proteinconcentration was assayed using BCA assay (Pierce).

Polyclonal Antibody Production and Purification

Purified partial murine recombinant pp32 protein as above was used toprime and boost two Pasteurella-free New Zealand rabbits using bi-weekly300 μg injections in Freund's adjuvant to produce polyclonal antibodies.For affinity purification of anti-recombinant pp32 from the rabbitserum, an affinity column was prepared and run as previously describedfor native pp32 (see Example 4).

Production of Human pp32 in Baculovirus

FIG. 12 shows a parallel experiment performed with the full-lengthprotein product of the human cDNA expressed in baculovirus and purifiedfrom infected SF9 cells. pp32BAC is a recombinant baculovirus into whichwe subcloned full-length pp32 under the polyhedrin promoter using thepVL1393 transfer plasmid (PharMingen). SF9 cells were maintained inGrace's insect medium supplemented with 10% heat-inactivated fetalbovine serum and 0.05 mg/ml gentamicin at 27°. Initially, SF9 cells wereco-transfected with the transfer plasmid and Baculogold virus by calciumphosphate precipitation. The resultant pp32BAC stocks were amplifiedfrom a single pfu obtained by end-point dilution. For production ofrecombinant pp32, SF9 cells infected with pp32BAC were grown in two 1 lspinner cultures stirred at 80-90 rpm at 27°; cells were infected at thebeginning of log phase growth and collected on day 4 post-infection,after having reached a density of 1.5×10⁶ cells/ml. Cells were harvestedand pelleted at 1000×g at 4°, washed with 100 ml medium without serum,pelleted and lysed in ice-cold lysis buffer consisting of 20 mMTris-HCl, pH 7.5 at 4°, containing 1% Triton X-100, 1 mM EDTA, 10 mMsodium pyrophosphate, 2 mM sodium vanadate, 3 mM ATP, 50 mM NaF, 0.5 mMdiisopropylfluorophosphate, and 0.1 mM phenylmethylsulfonyl fluoride at2×10⁸ cells/ml with vortexing every 3 min over the course of a 15 minincubation. Cell debris was removed by centrifugation at 17,800×g for 20min at 4° and recombinant pp32 was filtered at 0.45 μ and 0.22 μ andpurified by anion exchange HPLC (1).

FIG. 12 shows a Western blot of a 10% SDS-PAGE gel which was split anddeveloped alternatively with (A) affinity-purified polyclonal antibodyto a recombinant murine pp32 fragment or (B) the affinity-purifiedpolyclonal antibodies to denatured pp35 which originally detected pp32(see Example 4). The figure shows: 4.5 μg recombinant human pp32, lanes1; 0.45 μg purified native pp32 from HL-60 cells, lanes 2; and 120 μgfrom murine A₂₀ total cell lysate protein, lanes 3. Panel A was exposedfor 35 sec., and panel B for 5 min. Lane 3B shows the detection of bothpp35 and pp32 in A₂₀ lysate.

The purified recombinant human polypeptide (lanes 1) co-migrates withnative human pp32 purified from HL-60 cells (lanes 2) and with murinepp32 detected in total lysates of A₂₀ cells (lanes 3). Since the humanpp32 sequence predicts a polypeptide of 28,585 Da, migration at 32,000Da on SDS-PAGE gels likely reflects a retardative influence of theacidic domain of pp32, which would be unlikely to bind SDS well. Allthree polypeptide species react with antibodies specific for pp32 (panelA) and with the original antibodies designated anti-pp35d (1) whichfirst detected the existence of pp32. The in vitro translation productof the full-length murine clone also co-migrates with pp32 (FIG. 13).

In Vitro Transcription and Translation

Plasmids containing cDNA were linearized and RNA transcripts weresynthesized using Promega's RiboMAX T7 polymerase. 3 μg of pp32 RNA wasin vitro translated using Promega's rabbit reticulocyte lysate using ³⁵S-cysteine (ICN) at 30° for 1 hour. 25 μl of lysate was loaded onto a10% SDS-PAGE gel which was run, transferred to nitrocellulose, andexposed overnight at -80°. A₂₀ lysate was run alongside to compare thesize of the in vitro translated products with proteins recognized byanti-pp35 antibody.

35S-labeled in vitro translation products and A₂₀ lysate wereelectrophoresed on a 10% Laemmli gel and transferred to nitrocellulose.FIG. 13 compares a ³⁵ S autoradiograph of the filter with thecorresponding chemiluminescent Western blot image. The figure shows: invitro translation products of sense RNA from the HL2 human pp32 clone,lane A; A₂₀ lysate probed with the same antibody recognizing both pp35and pp32 illustrated in FIG. 12, lane B; and in vitro translationproduct of anti-sense RNA from the HL2 human pp35 clone, lane C.

Finally, previously published studies show that polyclonal antibodiesraised to and affinity-purified with the expressed murine recombinantfragment (v.s.) preferentially react with pp32 in Western blots of A₂₀cell lysates (5). The confluence of size and immunologic data thussupport the identification of the murine and human cDNA clones as pp32.

EXAMPLE 14

Sequence Analysis of pp32 cDNA

Sequence data also indirectly supports the identification of the cDNAclones as pp32. Sequences were analyzed using GCG version 7 software.The underlined sequences in FIG. 10A are identical to peptidesindependently sequenced from purified native pp35 (see Examples 3 and11) and thus are likely to represent peptides common to both pp32 andpp35. This supposition is reasonable, given the close correspondencebetween the peptide maps of the two proteins and the strongcross-reactivity between antibodies raised against either pp32 or pp35.Furthermore, the peptide sequence between residues 138 and 151 closelycorresponds to the previously reported sequence from the major pp35phosphopeptide (see Example 11); this same pp35 phosphopeptide sequencegave rise to the non-degenerate 42-base pair oligonucleotide probe usedto obtain the initial pp32 clones. The murine peptide and nucleotidesequences, shown in Table 3 below, are close but non-identical(differences are shown in bold) in pertinent ways. There is sufficientcorrespondence between the two nucleotide sequences to explain how thepp32 sequence was obtained with a pp35 probe. The pp35 sequence containsa serine at the position equivalent to the M¹⁴³ in the pp32 sequence; itis this serine which appears to be phosphorylated in murine pp35 bycasein kinase II (see Example 8). The absence of a casein kinase IIphosphorylation site in the pp32 peptide is consistent with theobservation that pp32 and pp35 phosphopeptides run distinctly from oneanother on reversed phase chromatography and precludes the possibilitythat the cDNA could encode pp35.

                  TABLE 3                                                         ______________________________________                                        pp35 Sequence                                                                           L L P Q L S Y L D G Y D D E, SEQ ID NO:6                            pp32 Sequence                                                                           L L P Q V M Y L D G Y D R D, SEQ ID NO:7                            Screening Probe                                                                         ctgctgccccagctgtcctacctggatggctatgatgatgag, SEQ ID                            NO:8                                                                pp32 Sequence                                                                           ctcctgccccaggtcatgtacctcgatggctatgacagggac, SEQ ID                            NO:9                                                                ______________________________________                                    

The sequence of pp32 predicts several interesting features, illustratedin FIG. 14, a cartoon of pp32 which illustrates the following features:P, potential casein kinase II phosphorylation sites; N, potentialnuclear localization signals; LVLL (human) and LILL (murine), potentialleucine zippers. Murine is apparently phosphorylated on a singlephosphoserine (Malek, et al., 1990); the phosphorylation state of humanpp32 has not yet been characterized.

Overall, pp32 is divided into two domains. The N-terminal two-thirds(residues 1 to 167) is generally amphipathic and has a high probabilityof alpha helical conformation according to Chou-Fasman predictions;approximately at its midpoint, the alpha helical domain has a potentialleucine zipper composed of leu-69, val-76, leu-83, and leu-90 (15). Theputative leucine zipper sequence is conserved in murine pp32 with onedifference: isoleucine is conservatively substituted for the secondvaline. These features suggest that pp32 might act throughself-association or through association with other molecules.Preliminary evidence obtained using size exclusion chromatographyindicates that both purified native human pp32 and purified full-lengthhuman pp32 produced in baculovirus exist in trimeric form in solution(Romantsev and Pasternack, unpublished observations). The N-terminalregion also contains two potential nuclear localization sequencescommencing at amino acids 61 and 108, respectively; each sequence iscomprised of a proline followed by a cluster of basic residues (16). TheC-terminal third of pp32 is highly acidic, composed of approximately 70%aspartic acid and glutamic acid residues; the predicted pI of human pp32is 3.81. While the function of this domain is presently unknown, similaracidic regions have been found on proteins as diverse as neurofilamenttriple helical L protein (17) and the major centromere binding protein,CENP-b (18).

Human and murine pp32 are quite homologous to one another, but noadditional close homologies can be found between pp32 and othermolecular species listed in the GenBank, EMBL, and SwissProt databases.Human and murine cDNA are 88% identical; the predicted proteins are 89%identical with conservative substitutions accounting for most of thedifferences and yielding 95% similarity. Molecules with acidic domainsare regularly detected due to the compositional monotony of the acidicregions. Similarly, neurofilament triple helical L protein is identifiedsince it shares the general organizational feature of an N-terminalamphipathic α-helix coupled with a C-terminal acidic tail. The oneexception concerns sequence X75090 (HSPHAPI), HLA-DR associated proteinI, submitted on Sep. 28, 1993 by M. Vaesen, S. Barnikol-Watanabe, H.Goetz, and N. Hilschmann of the Max Planck/Gottingen to GenBank. Thissequence is essentially identical to pp32. We have been unable toidentify a related published paper or abstract. We cannot comment on thesignificance of the findings or association with HLA-DR.

EXAMPLE 15

Expression of pp32Message

Human pp32 hybridizes with three RNA species in Northern blots ofpoly-A⁺ RNA from HL-60 cells, and hybridizes reciprocally with twomurine species from A₂₀ cells under highly stringent conditions. FIG. 15shows that similar RNA species are reciprocally identified in human andmurine RNA in blots probed with human and murine pp32 probes and washedat moderately high stringency.

Northern Blots

10 μg total RNA and 0.5 μg of poly-A+ mRNA from A₂₀ or HL-60's were runin formaldehyde/agarose gels using MOPS running buffer (8). RNA wastransferred to Amersham's Hybond nylon filters by capillary transfer in20×SSC. After overnight transfer, the blot was UV-crosslinked andhybridized using the same conditions for probing the HL-60 library withrandomly-primed probes. Filters were washed 2 times at room temperaturein 2×SSC and 2 times at 55° in 0.2×SSC. Autoradiography was performed at-80°.

Lanes A and C represent 10 μg of total human HL-60 RNA; lanes B and Drepresent 10 μg of total murine A₂₀ RNA. Lanes A and B were probed withhuman cDNA (clone HL2), and lanes C and D were probed with murine cDNA(clone 35.7). Lanes E and F represent 1 μg poly A⁺ human HL-60 RNAprobed either with the 5' HindIII fragment from clone HL2 (lane E) orwith an EcoRI fragment from HL13 representing the extended 3'untranslated sequences. All filters were washed at 55° in 0.2×SSC.

FIG. 15 shows that when the entire coding sequence is used as a probe, a1.3 kb species apparently corresponding to the cloned species isidentified, as well as additional species at 2.2 and 2.3 kb (lane A).The explanation for this phenomenon likely lies in alternative 3'polyadenylation signals, since one additional human pp32 mRNA specieshas been identified which is 881 nucleotides longer at its 3' end thanthe HL.2 clone shown in FIG. 10. Clone HL.13 is a 1766 bp cDNA fragmentfrom HL-60 cells which begins on nucleotide 176 of the HL.2 clone ofpp32 (FIG. 10) and extends for an additional 881 nucleotides beyond itstermination; no poly-A tail is present in HL.13 cDNA, although the 3'extension hybridizes with poly-A⁺ RNA. Except for the 3' extension, itis completely identical to the pp32 sequence. The 1.3, 2.2, and 2.3 kbmessages all hybridize in blots of human poly-A⁺ RNA probed with a 5'HindIII fragment of HL.2 (lane E) , whereas only the 2.2 and 2.3 kbmessages hybridize with an EcoRI fragment representing the 3' terminal273 bp of HL.13 (lane F). Based upon its hybridization pattern, it isprobable that the 2.3 kb species also represents a polyadenylationvariant. In human tissues and cell lines, the three pp32 mRNA speciesappear by Northern analysis to be roughly coequal in their expression.In contrast, only 1.3 and 2.2 kb species are seen in murine cell linessuch as A₂₀, with the 1.3 kb species predominating (lane D). Thepredominance of the 1.3 kb form may account for why no murine cDNAclones with extended 3' untranslated regions have been encountered sofar.

EXAMPLE 16

pp32 mRNA during Differentiation

p32 mRNA is subject to regulation in some cell lines. HL-60 cells are ahuman leukemic cell line which grow well in culture in anundifferentiated state. When exposed to certain agents such as phorbolester, HL-60 cells become adherent, express macrophage phenotypicmarkers, cease proliferation, and accumulate in G_(o) (19). FIG. 16shows that steady-state levels of pp32 mRNA greatly diminish when HL-60cells are exposed to TPA. In this experiment, HL-60 cells were incubatedwith 100 ng/ml TPA in DMSO or with DMSO vehicle for three hours, thenwashed and plated for incubation and further observation. 4×10⁵ HL-60cells/ml in 100 ml medium were incubated for 3 h at 37° with either 100ng tetradecanoyl phorbol acetate/ml (TPA, Gibco/BRL) from a 1 mg/mlstock in dimethylsulfoxide or an equal volume of dimethylsulfoxide. Ineach case, the final concentration of DMSO was 0.01%. The cells werethen harvested by centrifugation and either processed for total RNAextraction or washed twice in serum free medium and replated at 2.5×10⁶cells/ml in TPA-free medium. On successive days, cells were harvested bytrypsinization and processed for RNA (12).

HL-60 cells were induced to differentiate with TPA and total RNA wasextracted 3 hours, 1 day, 2 days, and 3 days after induction. Lanesmarked C on FIG. 16 represent control total RNA from HL-60 cellsincubated with DMSO vehicle alone. Each lane represents 10 μg total RNA.The blot was probed with human cDNA (HL2), then stripped and reprobedwith GAPDH cDNA as a loading control.

FIG. 16 shows a progressive decline of pp32 mRNA species relative to theGAPDH control observable by the first day following plating. By thethird day, virtually no pp32 message is present. Essentially identicalresults have been obtained in a system utilizing the induceddifferentiation of ML-1 cells (20, and data not shown). In the HL-60system, pp32 mRNA virtually disappears during the course ofdifferentiation; in a variety of systems, similar behavior has beennoted for p53 (21), myc (22,23), myb (20), and heat shock cognate 70(hsc70) (24). As an additional interesting feature, there is also amoderate diminution of pp32 mRNA in control cells incubated for the fullthree days; this may reflect a slight, suboptimal induction ofdifferentiation by the 0.01% DMSO used as a vehicle control (11).

The modulation of pp32 mRNA levels as a function of differentiation inHL-60 cells are entirely consistent with observations made in vivo thatpp32 mRNA levels high in self-renewing cell populations, but absent interminally differentiated cells. The experiments do not, however,establish the mechanism whereby pp32 mRNA levels are regulated. On onehand, it is possible that the results reflect modulation of pp32transcriptional activity; on the other hand, it is possible that pp32mRNA levels are regulated through message stability.

EXAMPLE 17

pp32 Inhibits ras-myc Transformation

In normal tissues in vivo, pp32 is selectively expressed in those cellscapable of self-renewal, which, from a teleological standpoint, shouldresist transformation. By this reasoning, pp32 might potentially act tosuppress one or more events leading to transformation. A countervailingobservation leads to precisely the opposite suggestion. pp32 is highlyexpressed in at least several forms of human neoplasia includingprostate cancer (5) and non-Hodgkin's lymphoma (Kuhajda and Pasternack,unpublished observations) at cellular levels and in proportions of cellswhich seem to increase with increasing severity of clinical disease.These data suggest that increased pp32 expression favors tumorigenesis.To begin to resolve these questions, we tested the effects of increasedpp32 expression on the well-characterized system of transformation ofrat embryo fibroblasts by ras and myc (25).

Transfection of Rat Embryo Fibroblasts

Primary rat embryo fibroblasts were either purchased from BioWhittakeror obtained courtesy of Dr. Chi Dang. For each experiment, approximately1×10⁶ cells were plated in T75 flasks and incubated for 2 d prior totransfection. For each flask of primary rat embryo fibroblasts, 5 μgpEJ-ras, and/or, 10 μg pMLV-c-myc, and/or 10 μg of pCMV32 or pSV32 andtwo volumes Lipofectin (twice the total μg DNA=μl Lipofectin) weregently mixed by inversion in 1.5 ml OPTIMEM in sterile 15 ml polystyrenetubes and allowed to incubate at room temperature for >15 min. Theamounts were increased proportionately when more than 1 flask was usedfor each transfection experiment. Cells were washed twice with sterilephosphate-buffered saline, once with OPTIMEM (Gibco-BRL), and then fedwith 6 ml of OPTIMEM and 1.5 ml of the DNA/Lipofectin mix. Afterovernight incubation, the cells were grown in standard media and refedwith fresh media twice weekly. Colonies were counted 2 weekspost-transfection. To determine the dose-dependence on pCMV32, pCMVvector and pCMV32 DNA were transfected such that the sum of the two wasa constant 12 μg. Ratios of 0:12, 3:9, 6:6, 9:3, and 12:0 μg pCMV:μgpCMV32 were cotransfected into triplicate flasks and foci were counted 2weeks later.

To determine whether any of the plasmids used were toxic, rat embryofibroblasts were co-transfected with pCMV, pCMV32, pSV40, or pSV32 alongwith pHyg. If any of the pp32-containing constructs were lethal, thenumber of hygromycin-resistant colonies from cells transfected with itwould be significantly less than control blank-plasmid transfectedcells. 10 μg of test vector and 1 μg of pHyg were transfected per flaskof cells using the protocol described above. Each transfection was donein duplicate. 48 h after transfection, 12.5 μg/ml of hygromycin wasadded to the medium and the cells were allowed to grow for one month,with a weekly medium change. Resistant colonies were stained withcrystal violet and counted.

Rat embryo fibroblasts were co-transfected with pEJ-ras, pMLV-c-myc, andthe indicated mixtures of pCMV32 and pCMV DNA such that the total amountof transfected DNA was constant in all samples. Colonies counted at 2weeks post transfection. The figure shows the mean of triplicatemeasurements±standard error.

FIG. 17 shows that pp32 inhibits ras-myc transformation of rat embryofibroblasts in a dose-dependent fashion. In this experiment, constantamounts of pEJras, pMLVmyc, and pCMV32+pCMV vector DNA were transientlyco-transfected into rat embryo fibroblasts. By three weekspost-transfection, cells treated with ras, myc, and 100% pCMV vector DNAproduced an average of nearly 100 colonies per flask; cells transfectedwith ras, myc and 100% pCMV32 DNA averaged around 20 colonies per flask.Intermediate fractions of pCMV32 DNA yielded intermediate dose-dependentreductions. Controls in which either ras or myc but not both weretransfected gave <1 average colony per flask.

The inhibition of ras-myc transformation by pp32 is highly reproducible,and is independent of both vector and promoter since similar resultswere obtained in constructs using an SV40 promoter (Table 4). The effectof pp32 cannot be explained by toxicity of the construct.Co-transfection of pCMV32 or pSV32 plasmids with thehygromycin-selectable plasmid pHyg (26) did not reduce the number ofdually hygromycin-resistant stable transfectants over those obtainedwith pCMV or pSV40 vectors alone (data not shown).

                  TABLE 4                                                         ______________________________________                                        pp32-Mediated Inhibition of ras- and                                          myc-Medicated Transformation is Independent of the Promoter                              FLASK                                                              Experiment                                                                            Plasmid  I      II  III   IV   % INHIBITION                           ______________________________________                                        I       pCMV     49     44  48    ND   70.9                                           pC32     8      15  18    ND                                          II      pCMV     42     52  47    ND   63.1                                           pC32                                                                  III     pSV40    25     24  20    28   73.2                                           pSV32    6      6   5     9                                           CONTROL NONE     43     47  ND    ND                                                  pCMV     48     37  ND    ND   5.6                                            pSV40    43     40  ND    ND   7.8                                    ______________________________________                                         Rat embryo fibroblasts were transfected with pEIras and pMLVc-myc with        either blank plasmids (pCMV or pSV40) or human pp32 cDNA (HL2) carrying       plasmids (pCMV32 or pSV32). Transformed foci were counted 2 weeks             posttransfection. Control experiments were performed to show that blank       plasmids had no effect on transformation (see text).                     

EXAMPLE 18

pp32 and bcl2 Both Cause Persistent Resistance to Drug-InducedProgrammed Cell Death

AT3 rat prostatic carcinoma cells stably transfected with pp32 areresistant to drug-induced programmed cell death (FIG. 18). In theseexperiments, AT3-pp32 cells were treated with ionomycin, thapsigargin,or 5-fluorouracil in dose- and time-dependent fashions, and theproportion of surviving cells determined. Death via apoptosis wasconfirmed by pulsed-field gel electrophoretic analysis ofdouble-stranded DNA breaks and by enumeration of apoptotic bodies. Thefigure shows the time course of 5-fluorouracil treatment in comparisonto AT3 cells stably transfected with bcl2, a cytoplasmic proteinconferring resistance, or to control cells.

For each assay, 3×10⁶ cells were plated in the presence or absence of0.1 μM 5-fluorodeoxyuridine. At the indicated times, cells wereharvested and counted. Programmed cell death was verified by pulsedfield gel electrophoretic analysis of double-strand DNA breaks. Δ, AT3cells with no drug treatment: ⋄, AT3-bcl2 clone 3 or AT3-pp32 clone 3;∘, AT3-bcl2 clone 4 or AT3-pp32 clone 5; □, AT3-neo. The figure showsthat stably transformed bcl2 and pp32 clones are both persistentlyresistant to drug-induced programmed cell death.

Programmed cell death requires induction, which can occur throughvarious signaling pathways, and execution, which likely occurs through afinal common pathway. 5-fluorouracil induces programmed cell death via aproliferation-dependent pathway, whereas ionomycin and thapsigargin donot, suggesting that induction occurs through different signalingpathways. The fact that pp32 inhibits both strongly suggests that itacts at the heart of the programmed cell death mechanism, the finallycommon pathway.

EXAMPLE 19

pp32Modulates Nuclear Morphology

pp32 induces changes in nuclear morphology suggestive of high-grademalignancy. FIG. 19 shows Papanicolaou-stained SF9 cells four daysfollowing infection with Baculovirus encoding pp32 (A), baculovirusencoding single-strand DNA binding protein (B), wild-type baculovirus(D), or nothing (C). Cells in C show diffuse, finely stippled nuclearchromatin with regular nuclei; the cells in D are similar, save forviral particles. The cells in B show changes common to overexpression ofmany nuclear proteins; there are non-specific nuclear inclusions, andsome of the nuclei are glassy. Similar changes occur with myc, whereasandrogen receptor has little effect. In contrast, pp32 in A producesenlarged, irregular nuclei with areas of coarsely clumped chromatin aswell as areas of clearing. Non-specific inclusions are also seen. Thepp32-specific changes are a cytologic hallmark of malignancy and do notappear to be a general feature of overexpression of a nuclear protein.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. Modificationsof the above-described modes for carrying out the invention that areobvious to persons of skill in medicine, immunology, hybridomatechnology, pharmacology, and/or related fields are intended to bewithin the scope of the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1052 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 97..843                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCCCAAAGTCCTAAAACGCGCGGCCGTGGGTTCGGGGTTTATTGATTGAATTCCGC60                CGGCGCGGGAGCCTCTGCAGAGAGAGAGCGCGAGAGATGGAGATGGGCAGACGG114                     MetGluMetGlyArgArg                                                            15                                                                            ATTCATTTAGAGCTGCGGAACAGGACGCCCTCTGATGTGAAAGAACTT162                           IleHisLeuGluLeuArgAsnArgThrProSerAspValLysGluLeu                              101520                                                                        GTCCTGGACAACAGTCGGTCGAATGAAGGCAAACTCGAAGGCCTCACA210                           ValLeuAspAsnSerArgSerAsnGluGlyLysLeuGluGlyLeuThr                              253035                                                                        GATGAATTTGAAGAACTGGAATTCTTAAGTACAATCAACGTAGGCCTC258                           AspGluPheGluGluLeuGluPheLeuSerThrIleAsnValGlyLeu                              404550                                                                        ACCTCAATCGCAAACTTACCAAAGTTAAACAAACTTAAGAAGCTTGAA306                           ThrSerIleAlaAsnLeuProLysLeuAsnLysLeuLysLysLeuGlu                              55606570                                                                      CTAAGCGATAACAGAGTCTCAGGGGGCCTAGAAGTATTGGCAGAAAAG354                           LeuSerAspAsnArgValSerGlyGlyLeuGluValLeuAlaGluLys                              758085                                                                        TGTCCGAACCTCACGCATCTAAATTTAAGTGGCAACAAAATTAAAGAC402                           CysProAsnLeuThrHisLeuAsnLeuSerGlyAsnLysIleLysAsp                              9095100                                                                       CTCAGCACAATAGAGCCACTGAAAAAGTTAGAAAACCTCAAGAGCTTA450                           LeuSerThrIleGluProLeuLysLysLeuGluAsnLeuLysSerLeu                              105110115                                                                     GACCTTTTCAATTGCGAGGTAACCAACCTGAACGACTACCGAGAAAAT498                           AspLeuPheAsnCysGluValThrAsnLeuAsnAspTyrArgGluAsn                              120125130                                                                     GTGTTCAAGCTCCTCCCGCAACTCACATATCTCGACGGCTATGACCGG546                           ValPheLysLeuLeuProGlnLeuThrTyrLeuAspGlyTyrAspArg                              135140145150                                                                  GACGACAAGGAGGCCCCTGACTCGGATGCTGAGGGCTACGTGGAGGGC594                           AspAspLysGluAlaProAspSerAspAlaGluGlyTyrValGluGly                              155160165                                                                     CTGGATGATGAGGAGGAGGATGAGGATGAGGAGGAGTATGATGAAGAT642                           LeuAspAspGluGluGluAspGluAspGluGluGluTyrAspGluAsp                              170175180                                                                     GCTCAGGTAGTGGAAGACGAGGAGGACGAGGATGAGGAGGAGGAAGGT690                           AlaGlnValValGluAspGluGluAspGluAspGluGluGluGluGly                              185190195                                                                     GAAGAGGAGGACGTGAGTGGAGAGGAGGAGGAGGATGAAGAAGGTTAT738                           GluGluGluAspValSerGlyGluGluGluGluAspGluGluGlyTyr                              200205210                                                                     AACGATGGAGAGGTAGATGACGAGGAAGATGAAGAAGAGCTTGGTGAA786                           AsnAspGlyGluValAspAspGluGluAspGluGluGluLeuGlyGlu                              215220225230                                                                  GAAGAAAGGGGTCAGAAGCGAAAACGAGAACCTGAAGATGAGGGAGAA834                           GluGluArgGlyGlnLysArgLysArgGluProGluAspGluGlyGlu                              235240245                                                                     GATGATGACTAAGTGGAATAACCTATTTTGAAAAATTCCTATTGTGATT883                          AspAspAsp                                                                     TGACTGTTTTTACCCATATCCCCTCTCCCCCCCCCCTCTAATCCTGCCCCCTGAAACTTA943               TTTTTTTCTGATTGTAACGTTGCTGTGGGAACGAGAGGGGAAGAGTGTACTGGGGGTTGC1003              GGGGGGAGGATGGCGGGTGGGGGTGGAATAAAATACTATTTTTACTGCC1052                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 249 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluMetGlyArgArgIleHisLeuGluLeuArgAsnArgThrPro                              151015                                                                        SerAspValLysGluLeuValLeuAspAsnSerArgSerAsnGluGly                              202530                                                                        LysLeuGluGlyLeuThrAspGluPheGluGluLeuGluPheLeuSer                              354045                                                                        ThrIleAsnValGlyLeuThrSerIleAlaAsnLeuProLysLeuAsn                              505560                                                                        LysLeuLysLysLeuGluLeuSerAspAsnArgValSerGlyGlyLeu                              65707580                                                                      GluValLeuAlaGluLysCysProAsnLeuThrHisLeuAsnLeuSer                              859095                                                                        GlyAsnLysIleLysAspLeuSerThrIleGluProLeuLysLysLeu                              100105110                                                                     GluAsnLeuLysSerLeuAspLeuPheAsnCysGluValThrAsnLeu                              115120125                                                                     AsnAspTyrArgGluAsnValPheLysLeuLeuProGlnLeuThrTyr                              130135140                                                                     LeuAspGlyTyrAspArgAspAspLysGluAlaProAspSerAspAla                              145150155160                                                                  GluGlyTyrValGluGlyLeuAspAspGluGluGluAspGluAspGlu                              165170175                                                                     GluGluTyrAspGluAspAlaGlnValValGluAspGluGluAspGlu                              180185190                                                                     AspGluGluGluGluGlyGluGluGluAspValSerGlyGluGluGlu                              195200205                                                                     GluAspGluGluGlyTyrAsnAspGlyGluValAspAspGluGluAsp                              210215220                                                                     GluGluGluLeuGlyGluGluGluArgGlyGlnLysArgLysArgGlu                              225230235240                                                                  ProGluAspGluGlyGluAspAspAsp                                                   245                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 980 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: mus sp                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGCACGAGAAGAGAGAGCGCGAGAGATGGAGATGGACAAACGGATTTATTTAGAGCTGCG60                GAACAGGACGCCCTCTGATGTGAAAGAGCTGGTCCTGGATAACTGTAAGTCAATTGAAGG120               CAAAATCGAAGGCCTCACGGATGAGTTTGAAGAACTGGAATTCCTAAGTACAATCAACGT180               AGGCCTCACCTCCATTTCCAACTTACCAAAGTTAAACAAACTCAAGAAGCTTGAATTAAG240               CGAAAACAGAATCTCAGGGGACCTGGAAGTATTGGCAGAGAAATGTCCGAACCTTAAGCA300               TCTAAATTTAAGTGGCAACAAAATAAAAGATCTCAGCACAATAGAGCCGCTGAAGAAGTT360               AGAGAATCTCAAGAGCCTAGACCTGTTTAACTGTGAGGTGACCAACCTGAATGCCTACCG420               AGAAAACGTGTTCAAGCTCCTGCCCCAGGTCATGTACCTCGATGGCTATGACAGGGACAA480               CAAGGAGGCCCCCGACTCCGATGTTGAGGGCTACGTGGAGGATGACGACGAGGAAGATGA540               GGATGAGGAGGAGTATGATGAATATGCCCAGCTAGTGGAAGATGAAGAGGAAGAGGTTGA600               GGAGGAAGAAGGGGAGGAAGAGGATGTGAGTGGAGAGGAGGAGGAGGATGAGGAAGGTTA660               CAATGACGGGGAAGTGGATGACGAGGAAGACGAAGAAGAAGCTGGTGAAGAAGAAGGGAG720               TCAGAAGCGAAAACGAGAACCGGACGATGAGGGCGAAGAGGATGACTAAGGAATGAACCT780               GTTTGGGGAAATTCCTATTGTGATTTGACTGTTTTTACCCATATCCCCTCCCCCTCCTAT840               TCCTGCCCCCCGAAACTTATTTTTTTCTGATTGTAGCATTGCTGTGGGAAGGAGAGGGGA900               AAAGTGTACTGGGGGTTGATGGGGGGTGGGGGTGGGGGGGAGGGGTGGAATAAAATACTA960               TTTTTACTGCCACACTTTAC980                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 759 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus sp                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..548                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGGTCAAGAAGCTTGAATTAAGCGAAAACAGAATCTCAGGGGACCTG47                             ValLysLysLeuGluLeuSerGluAsnArgIleSerGlyAspLeu                                 151015                                                                        GAAGTATTGGCAGAGAAATGTCCGAACCTTAAGCATCTAAATTTAAGT95                            GluValLeuAlaGluLysCysProAsnLeuLysHisLeuAsnLeuSer                              202530                                                                        GGCAACAAAATAAAAGATCTCAGCACAATAGAGCCGCTGAAGAAGTTA143                           GlyAsnLysIleLysAspLeuSerThrIleGluProLeuLysLysLeu                              354045                                                                        GAGAATCTCAAGAGCCTAGACCTGTTTAACTGTGAGGTGACCAACCTG191                           GluAsnLeuLysSerLeuAspLeuPheAsnCysGluValThrAsnLeu                              505560                                                                        AATGCCTACCGAGAAAACGTGTTCAAGCTCCTGCCCCAGGTCATGTAC239                           AsnAlaTyrArgGluAsnValPheLysLeuLeuProGlnValMetTyr                              657075                                                                        CTCGATGGCTATGACAGGGACAACAAGGAGGCCCCCGACTCCGATGTT287                           LeuAspGlyTyrAspArgAspAsnLysGluAlaProAspSerAspVal                              80859095                                                                      GAGGGCTACGTGGAGGATGACGACGAGGAAGATGAGGATGAGGAGGAG335                           GluGlyTyrValGluAspAspAspGluGluAspGluAspGluGluGlu                              100105110                                                                     TATGATGAATATGCCCAGCTAGTGGAAGATGAAGAGGAAGAGGTTGAG383                           TyrAspGluTyrAlaGlnLeuValGluAspGluGluGluGluValGlu                              115120125                                                                     GAGGAAGAAGGGGAGGAAGAGGATGTGAGTGGAGAGGAGGAGGAGGAT431                           GluGluGluGlyGluGluGluAspValSerGlyGluGluGluGluAsp                              130135140                                                                     GAGGAAGGTTACAATGACGGGGAAGTGGATGACGAGGAAGACGAAGAA479                           GluGluGlyTyrAsnAspGlyGluValAspAspGluGluAspGluGlu                              145150155                                                                     GAAGCTGGTGAAGAAGAAGGGAGTCAGAAGCGAAAACGAGAACCGGAC527                           GluAlaGlyGluGluGluGlySerGlnLysArgLysArgGluProAsp                              160165170175                                                                  GATGAGGGCGAAGAGGATGACTAAGGAATGAACCTGTTTGGGGAAATTCCT578                        AspGluGlyGluGluAspAsp                                                         180                                                                           ATTGTGATTTGACTGTTTTTACCCATATCCCCTCCCCCTCCTATTCCTGCCCCCCGAAAC638               TTATTTTTTTCTGATTGTAGCATTGCTGTGGGAAGGAGAGGGGAAAAGTGTACTGGGGGT698               TGATGGGGGGTGGGGGTGGGGGGGAGGGGAATAAAATACTATTTTTACTGCCACACTTTA758               C759                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ValLysLysLeuGluLeuSerGluAsnArgIleSerGlyAspLeuGlu                              151015                                                                        ValLeuAlaGluLysCysProAsnLeuLysHisLeuAsnLeuSerGly                              202530                                                                        AsnLysIleLysAspLeuSerThrIleGluProLeuLysLysLeuGlu                              354045                                                                        AsnLeuLysSerLeuAspLeuPheAsnCysGluValThrAsnLeuAsn                              505560                                                                        AlaTyrArgGluAsnValPheLysLeuLeuProGlnValMetTyrLeu                              65707580                                                                      AspGlyTyrAspArgAspAsnLysGluAlaProAspSerAspValGlu                              859095                                                                        GlyTyrValGluAspAspAspGluGluAspGluAspGluGluGluTyr                              100105110                                                                     AspGluTyrAlaGlnLeuValGluAspGluGluGluGluValGluGlu                              115120125                                                                     GluGluGlyGluGluGluAspValSerGlyGluGluGluGluAspGlu                              130135140                                                                     GluGlyTyrAsnAspGlyGluValAspAspGluGluAspGluGluGlu                              145150155160                                                                  AlaGlyGluGluGluGlySerGlnLysArgLysArgGluProAspAsp                              165170175                                                                     GluGlyGluGluAspAsp                                                            180                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus sp                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuLeuProGlnLeuSerTyrLeuAspGlyTyrAspAspGlu                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus sp                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuLeuProGlnValMetTyrLeuAspGlyTyrAspArgAsp                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: YES                                                       (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CTGCTGCCCCAGCTGTCCTACCTGGATGGCTATGATGATGAG42                                  (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus sp                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTCCTGCCCCAGGTCATGTACCTCGATGGCTATGACAGGGAC42                                  __________________________________________________________________________

I claim:
 1. A preparation of antibodies which specifically binds to a amammalian protein comprising an amino acid sequence as shown in SEQ IDNO:
 5. 2. An antibody which specifically binds a human nuclear proteinin native conformation, said human protein being purified from humancells by lysing the cells in hypotonic medium containing non-ionicdetergent and salt concentration from 5-50 mM, recovering a lysate, andfractionating the lysate by multi-stage anion exchange chromatography,said human protein having a molecular weight of about 32 kDa as measuredby SDS-PAGE under denaturing conditions, said human protein beingdetected in Western blot by affinity-purified polyclonal antibodieswhich specifically bind a murine protein in native conformation, saidmurine protein containing the amino acid sequence of SEQ ID NO:
 5. 3.The antibody according to claim 1, wherein the antibody is in anisolated polyclonal antiserum, a preparation of purified polyclonalantibodies, or a preparation containing one or more monoclonalantibodies.
 4. A preparation of antibodies which specifically binds to amammalian protein with an amino acid sequence encodes by SEQ ID NO: 1.5. The antibody according to claim 4, wherein the antibody is in anisolated polyclonal antiserum, a preparation of purified polyclonalantibodies, or a preparation containing one or more monoclonalantibodies.